˘ ˇˆ˙ - Biblioteca UEXbiblioteca.unex.es/tesis/9788469263174.pdf · mayor frecuencia en...

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INTRODUCCIÓN

1

I. INTRODUCCIÓN

I.1. POBLACIÓN FÚNGICA EN ALIMENTOS

Las condiciones ambientales en que se procesan o se almacenan diversos tipos de

alimentos favorecen la proliferación en su superficie de mohos que tienen la capacidad de

adaptarse y desarrollarse en sustratos muy diversos. Las especies de mohos aisladas con

mayor frecuencia en alimentos pertenecen a los géneros Penicillium, Aspergillus,

Fusarium y Eurotium, aunque las características intrínsecas de cada tipo de producto

condicionan el desarrollo de unas especies u otras (Filtenborg y col., 1996). Así, frutas y

verduras frescas suelen alterarse durante su almacenamiento por P. expansum, P.

crustosum, P. solitum, P. digitatum o P. italicum (Pitt y Hocking, 1997; Moss, 2008), y los

cereales en cambio pueden ser colonizados por P. polonicum, P. verrucosum, P.

aurantiogriseum, P. viridicatum, A. flavus, A. niger o A. versicolor (Filtenborg y col.,

1996).

En productos cárnicos madurados y quesos es frecuente el desarrollo una población

fúngica superficial durante la maduración que puede tener efectos deseables, ya que

contribuye en gran medida a la formación del sabor, aroma o textura del producto acabado

(Kinsella y Hwang, 1976; Martín y col., 2004, 2006), e incluso puede ejercer un efecto

protector frente a otros microorganismos no deseados (Geisen y col., 1992; Leistner, 1994;

Singh y Dincho, 1994; Berwal y Dincho, 1995; Molimard y col., 1995; Decker y Nielsen,

2005). Por ello, en quesos es frecuente el uso de determinadas cepas de mohos como P.

roqueforti o P. camemberti como cultivos iniciadores (Nielsen y col., 1998), y se ha

propuesto la elaboración de este tipo de cultivos para su utilización en productos cárnicos

madurados a partir de cepas no toxigénicas de P. chrysogenum (Benito y col., 2005; Martín

y col., 2006), Penicillium olsonii (López-Díaz y col., 2001) o Penicillium camemberti

(Bruna y col., 2003).

Pero el desarrollo de mohos no siempre es deseable en alimentos. En quesos se han

descrito cepas alterantes de P. commune y P. nalgiovense que pueden conducir a un

deterioro en las características sensoriales (Filtenborg y col., 1996), debido normalmente a

la liberación de exoenzimas durante su desarrollo. Estos enzimas pueden continuar su

actividad independientemente de que se produzca la eliminación del micelio, causando

desde olores o coloraciones desagradables hasta la completa desintegración de la estructura

del alimento. No obstante, el aspecto más preocupante respecto del desarrollo de una

INTRODUCCIÓN

2

población fúngica incontrolada en alimentos es su capacidad para producir micotoxinas. El

desarrollo de mohos en quesos puede conllevar la producción de micotoxinas (Marth y

Yousef, 1991), muchas de las cuales han sido detectadas en estos productos (Lafont y col.,

1979; Le Bars, 1979; Northolt y col., 1980; Jarvis, 1983; Siemens y Zawistowski, 1993).

De manera similar, en productos cárnicos madurados la población microbiana superficial

está constituida fundamentalmente por mohos, muchos de los cuales son toxigénicos

(Bullerman y col., 1969; Escher y col., 1973; Halls y Ayres, 1973; Wu y col., 1974;

Leistner, 1984; Núñez y col., 1996; López-Díaz y col., 2001).

I.2. CONSECUENCIAS DEL DESARROLLO DE MOHOS EN PRODUCTOS CÁRNICOS

MADURADOS

Con respecto a la población fúngica que puede colonizar la superficie de productos

cárnicos, existen tanto mohos que contribuyen en gran medida a la formación del sabor,

aroma o textura del producto acabado, como especies que pueden deteriorar las

características sensoriales o producir micotoxinas.

I.2.1. EFECTOS BENEFICIOSOS

La población fúngica que coloniza los productos cárnicos madurados presenta

diversas actividades metabólicas que pueden mejorar las características sensoriales del

producto. Así, la presencia de mohos puede ofrecer actividad antioxidante, lipolítica,

proteolítica, contribuir al aroma y sabor del producto e incluso ejercer un efecto protector

por su actividad antimicrobiana (Figura I1).

El efecto antioxidante de los mohos se atribuye a que la mayoría de las cepas

aisladas de jamón poseen una actividad catalasa moderada (Núñez, 1995), destruyendo

peróxidos y reduciendo las reacciones oxidativas (Burgos, 1981), por lo que disminuye el

enranciamiento y contribuyen a la estabilización del color del producto (Liepe, 1982;

Smith y Palumbo, 1983; Geisen, 1993). Además, muchos mohos producen compuestos con

acción antioxidante (Ludemann y col., 2004).

Por otra parte, numerosas especies de mohos pertenecientes al género Penicillium

poseen exoenzimas con actividad lipolítica que pueden incrementar la concentración de

ácidos grasos libres y compuestos volátiles en jamón (Huerta y col., 1987; Molina y col.,

1991) y embutidos (Toledo y col., 1996), contribuyendo así al desarrollo del sabor y aroma

del producto. Se ha observado actividad lipolítica en mohos del género Penicillium

INTRODUCCIÓN

3

aislados de jamón ibérico (Núñez, 1995; Alonso, 2004) y de embutidos (Selgas y col.,

1999; Ludemann y col., 2004).

Adicionalmente, aunque la proteolisis en los productos cárnicos se ha asociado

generalmente a la actividad de los enzimas presentes en el tejido muscular (Toldrá y col.,

1992; Sárraga y col., 1993), algunas cepas aisladas de jamón curado producen una intensa

hidrólisis de proteínas miofibrilares y un gran incremento de aminoácidos libres cuando se

inoculan en carne de cerdo estéril y productos cárnicos madurados (Rodríguez y col., 1998;

Martín y col., 2001, 2002, 2004). En este sentido, se ha demostrado una intensa actividad

proteolítica de una cepa de P. chrysogenum (Rodríguez y col., 1998; Martín y col., 2001,

2002; Benito y col., 2003; Alonso, 2004), a partir de la cual se ha purificado una proteasa

muy activa en las condiciones de maduración de diferentes productos cárnicos (Benito y

col., 2002). También se han aislado de embutidos mohos muy proteolíticos de los géneros

Mucor y Penicillium (Trigueros y col., 1995), capaces de incrementar la concentración de

aminoácidos libres en estos productos (Bruna y col., 2001).

Los mohos pueden favorecer la formación del aroma característico de los productos

curados por diversas vías. En primer lugar, los aminoácidos liberados, además de potenciar

el sabor de los alimentos (Suyama y Shimuzu, 1982; Nishimura y Kato, 1988), pueden

Figura I1. Efectos beneficiosos por el desarrollo de mohos durante la maduración del

jamón (Núñez, 1995).

INTRODUCCIÓN

4

intervenir en reacciones de condensación de Maillard (Shahidi y col., 1986; Mottram y

Whitfield, 1995) y de degradación de Strecker (Barbieri y col., 1992), en las que se pueden

formar compuestos responsables del aroma del jamón curado (García y col., 1991; Ruiz y

col., 1998; Whitfield, 1992).

Finalmente, los mohos pueden tener un efecto protector frente a microorganismos

patógenos o alterantes (Geisen y col., 1992; Leistner, 1994; Singh y Dincho, 1994; Berwal

y Dincho, 1995). Así, el 75% de los mohos aislados en jamón ibérico inhiben el

crecimiento de Staphylococcus aureus (Núñez y col., 1996), y diversas especies no

toxigénicas de mohos tienen la capacidad de inhibir la producción de micotoxinas e incluso

de destruir las formadas por otras especies toxigénicas (Mislivec y col. 1988). No obstante,

este efecto podría deberse a actividades tan poco deseables como la producción de toxinas

o de antibióticos, o bien a mecanismos más deseables como a interferencias metabólicas o

a la liberación de proteínas antimicrobianas.

I.2.2. EFECTOS PERJUDICIALES

El desarrollo de ciertos mohos, como los géneros Mucor y Rhizopus, puede

provocar un aspecto no deseable al jamón o embutidos (Figura I2). Por otro lado, los

mohos con gran capacidad lipolítica pueden ocasionar olores anómalos (Sanz, 1986). De

hecho, se ha identificado a P. commune como el responsable del “defecto del ácido

fénico”, caracterizado por conferir al jamón un fuerte olor a fenol (Spotti y col., 1988).

Figura I2. Contaminación fúngica superficial en jamón curado.

INTRODUCCIÓN

5

No obstante, el aspecto más preocupante respecto al desarrollo de una población

fúngica superficial en estos productos es su capacidad para producir micotoxinas. Las

micotoxinas son metabolitos secundarios tóxicos que al ser ingeridos en pequeñas

cantidades, pueden ocasionar diferentes patologías relacionadas con inmunosupresión,

carcinogénesis, neurotoxicidad, etc. (Samson y col., 1995; Richard, 2007). Existen

micotoxinas que sólo son producidas por un grupo reducido de mohos, y otras generadas

por especies muy diversas incluso de géneros diferentes (Frisvad y Thrane, 1995). En este

sentido, se han descrito especies como Penicillium griseofulvum capaces de producir gran

variedad de micotoxinas (Frisvad y Filtenborg, 1990). No obstante, el perfil y la

concentración de micotoxinas que puede producir un moho, así como la posterior difusión

al interior del alimento, no sólo dependen de la naturaleza del microorganismo sino

también de la composición del alimento y del procesado al que esté sometido. Además,

estos metabolitos son muy resistentes a los tratamientos físicos y químicos, por lo que una

vez que las micotoxinas están en el alimento pueden permanecer en él incluso después del

procesado (Osborne y col., 1996; Castellá y col., 1998; Katta y col., 1999). Muchos mohos

colonizadores de alimentos son capaces además de producir antibióticos como la penicilina

(Andersen y Frisvad, 1994), lo que supone un riesgo para los consumidores alérgicos a este

antibiótico (Bremmelgaard, 1998).

El potencial toxigénico de los mohos aislados de productos cárnicos se ha

establecido en distintos trabajos. Se ha comprobado que el 80% de 1481 aislados del

género Penicillium a partir de embutidos son potencialmente toxigénicos (Eckardt y col.,

1979). Más del 75% de los mohos aislados en jamón ibérico muestran toxicidad o

mutagenicidad en ensayos biológicos (Núñez y col., 1996). Entre estos mohos, el más

frecuente es P. commune, productor de ácido ciclopiazónico (Núñez y col., 1996; Sosa y

col., 2002), destacando también P. restrictum, A. flavus y A. niger. Además, todos los

aislados de P. commune obtenidos de chorizo de Cantimpalos mostraron una alta toxicidad

para larvas de Artemia salina (López-Díaz y col., 2001). Estas especies pueden producir

micotoxinas en los productos cárnicos sobre los que se desarrollan (Núñez y col., 2007),

habiéndose comprobado en embutidos y jamones curados la presencia de citrinina,

patulina, rugulosina, ocratoxina A, ácido ciclopiazónico, citreoviridina, breviamida A,

fumitremorgina B, griseofulvina y verruculógeno TR1 (Leistner, 1984). Además, las

micotoxinas pueden difundir al interior de los productos, donde pueden mantenerse incluso

cuando el micelio se ha eliminado (Leistner, 1984).

INTRODUCCIÓN

6

Por todo esto, resulta preocupante el crecimiento incontrolado de mohos

potencialmente toxigénicos en la superficie de los productos cárnicos madurados (Núñez y

col., 1996; Sabater-Vilar y col., 2003) y sería recomendable controlar su desarrollo en este

tipo de productos.

I.3. CONTROL DE MOHOS EN PRODUCTOS CÁRNICOS MADURADOS

Existen numerosos mecanismos para controlar el desarrollo microbiano en distintos

tipos de alimentos. Entre los métodos físicos de control se encuentra la utilización de

tratamientos térmicos, radiaciones ionizantes o ultravioleta, altas presiones, pulsos

eléctricos o de luz, o campos magnéticos. También pueden emplearse agentes químicos,

incluyendo ácidos orgánicos, aceites esenciales, peróxido de hidrógeno, etc., así como el

envasado en atmósferas controladas o modificadas. Estos tratamientos resultan eficaces

para evitar el desarrollo de mohos en determinados alimentos, como frutas, verduras, pan,

etc. No obstante, en los productos cárnicos madurados está limitada la aplicación directa de

fungicidas. Además, las medidas higiénicas para prevenir la contaminación durante la

elaboración se ven desbordadas en los secaderos y bodegas, donde resulta muy difícil

eliminar totalmente la contaminación ambiental. Por otra parte, la inespecificidad de estos

tratamientos evitaría la posible contribución beneficiosa de los mohos a la maduración de

estos productos.

Por todo ello, en productos cárnicos madurados es necesario adoptar estrategias

alternativas que garanticen el desarrollo de los microorganismos deseables y la seguridad

de estos alimentos.

I.3.1. CULTIVOS PROTECTORES

Para controlar la población fúngica que se desarrolla en la superficie de los

productos cárnicos madurados podrían utilizarse cultivos protectores formados por mohos

capaces de inhibir por competición a los mohos no deseables, siempre teniendo en cuenta

que deben ser no toxigénicos y mostrar una actividad metabólica adecuada. No obstante,

esta opción se ve limitada por la evolución de las condiciones ecológicas durante la

maduración, resultando muy poco probable que los mohos seleccionados superen a todas

las especies habituales en la amplísima gama de temperatura y actividad de agua que tiene

lugar durante la maduración (Rodríguez y col., 1994). Una posibilidad radicaría en el

empleo de mohos que dispusieran de algún elemento adicional que favoreciese su

INTRODUCCIÓN

7

desarrollo, como la producción de antibióticos. Lógicamente, esta opción se ha descartado

por las posibles reacciones alérgicas en el consumidor o la inducción de resistencia en

otros microorganismos como consecuencia de su empleo continuado.

Otra posible estrategia consiste en el empleo de proteínas antibacterianas como las

bacteriocinas, que están siendo ampliamente utilizadas en otro tipo de alimentos, o de los

microorganismos que las producen. Así, la nisina es una bacteriocina autorizada como

conservante alimentario (Gálvez y col., 2007). Pero este tipo de proteínas, a pesar de tener

un espectro de inhibición muy amplio, no inhibe a los mohos. Por ello, ni las bacteriocinas

ni los microorganismos que las producen pueden ser empleados para evitar el crecimiento

fúngico en alimentos.

Sin embargo, la reciente descripción de proteínas antifúngicas naturales elaboradas

por mohos abre nuevas perspectivas en este sentido. La utilización de cepas de mohos

productoras de proteínas antifúngicas como cultivos protectores en productos cárnicos

madurados, permitiría limitar el desarrollo de mohos toxigénicos sin afectar a los

deseables, contribuyendo así a estandarizar las características del producto.

I.3.2. PROTEÍNAS ANTIFÚNGICAS

La investigación de nuevos agentes antifúngicos ha experimentado un notable

avance en los últimos años como consecuencia del incremento de presentación de micosis

en pacientes inmunodeprimidos por el SIDA o por tratamientos anticancerígenos,

especialmente infecciones por distintas especies de Candida o en aspergilosis invasivas

(Anaissie, 2008; de la Torre y col., 2008; Pemán y Almirante, 2008). Esta búsqueda de

nuevos compuestos antifúngicos se ha extendido también a otros campos como la

agricultura, donde la proliferación de mohos patógenos en los cultivos ocasiona grandes

pérdidas económicas. Así, se han caracterizado nuevas proteínas con actividad antifúngica

(Upadhyay y Srivastava., 2008; Yan y col., 2008) y se han producido plantas transgénicas

que las expresen (Girlanda y col., 2008; Swathi-Anyradha y col., 2008).

En los últimos años se han purificado y caracterizado proteínas con efecto

antifúngico a partir de plantas (Chu y col., 2003; Ng y col., 2003), bacterias (Yang y col.,

2002), mohos (Marx y col., 2007; Meyer, 2008), artrópodos (Tomie y col., 2003), o

reptiles (Gomes y col., 2005), así como también se han diseñado péptidos sintéticos

(Patrzykaty col., 2003).

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El mecanismo de acción de las proteínas antifúngicas es tan variado como los

organismos que las producen (Selitrennikoff, 2001; Theis y Stahl, 2004). Algunos

atraviesan la membrana e interfieren en la síntesis de ADN, ARN o de proteínas, inhiben la

cadena respiratoria celular (Cociancich y col., 1993), disminuyen el ATP (Cociancich y

col., 1993), inactivan los ribosomas fúngicos (Hwu y col., 2000) o provocan la

desporalización de la membrana (Thevissen y col., 1997). Otros en cambio forman canales

y poros en la membrana alterando su permeabilidad y provocando la pérdida de iones y

otros solutos (Thevissen y col., 1999; Lee y col., 2001) o pueden interferir en la síntesis de

componentes esenciales de la pared celular como el �-1,3-glucano (Odds y col., 2003), la

quitina (Yang y Gong, 2002; Von der Weid y col., 2003) o el quitosán (Shimosaka y col.,

2005), pudiendo incluso hidrolizarlos (Sela-Buurlage y col., 1993; Graham y Sticklen,

1994). Esto debilita la pared y origina la lisis de la célula.

No obstante, las proteínas antifúngicas de mayor utilidad son las que presentan

mecanismos de acción específicos para la cubierta externa de los hongos (Groll y col.,

1998), ya que suelen ser poco tóxicas para las células de mamíferos. En la interacción de la

proteína con la membrana desempeñan un papel destacado la carga (Hoover y col., 2003) y

el carácter catiónico o hidrofóbico del compuesto (Friedrich y col., 1999; Hancock y

Chapple, 1999). Así, la hidrofobicidad y la estructura en �-hélice, se correlacionan con la

toxicidad para las células de mamíferos, mientras que la carga positiva está más

relacionada con la actividad antimicrobiana (Hong y col., 2001). Esto se debe a que las

membranas celulares de bacterias y mohos se componen principalmente de fosfolípidos

aniónicos que favorecen la unión de la proteína antifúngica y su actividad, mientras que

estos fosfolípidos no están presentes en muchos eucariotas superiores o están confinados

en la cara interna de la membrana plasmática, no estando en contacto con el medio

extracelular (Rivas y Andreu, 2003; Theis y Stahl, 2004).

Por otra parte, existe una gran variabilidad en cuanto a la composición de

aminoácidos y estructura secundaria y terciaria de las proteínas antifúngicas, aunque la

mayoría se caracterizan por ser catiónicas y por formar estructuras anfipáticas (Hwang y

Vogel, 1998). Se han descrito cuatro proteínas antifúngicas catiónicas y de bajo peso

molecular producidas por mohos: AFP de Aspergillus giganteus (Nakaya y col., 1990;

Lacadena y col., 1995), PAF de Penicillium chrysogenum (Marx y col., 1995), Anafp de

Aspergillus niger (Lee y col., 1999) y AcAFP de A. clavatus (Skouri-Gargouri y Gargouri,

2008). Todas ellas se caracterizan por su pequeño tamaño (51-58 aminoácidos), su alto

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contenido en residuos de cisteína y su fuerte carácter básico. Además, sus estructuras

terciarias están estabilizadas por tres o cuatro puentes disulfuro y forman estructuras con

un marcado carácter anfipático (Nakaya y col., 1990; Lacadena y col., 1995).

Sin embargo, la aplicación de los mohos productores como cultivos protectores en

alimentos madurados en general y en productos cárnicos en particular exige que dichos

mohos estén adaptados a las condiciones ecológicas de estos alimentos, que no los alteren

y que no sean productores de micotoxinas. Ninguna de las cepas a partir de las que se han

purificado proteínas antifúngicas ha sido obtenida a partir de productos madurados. Por

ello, es esperable que no se adapten a las condiciones ecológicas particulares de este tipo

de productos de manera que puedan competir con los mohos que crecen de forma

espontánea. Además, algunos de estos mohos pertenecen al género Aspergillus, que sólo es

predominante en estos alimentos en las últimas fases de su elaboración/maduración,

cuando la aw es muy baja. Por lo tanto, la utilización de mohos requiere encontrar cepas

adaptadas a este tipo de alimentos y que sean capaces de elaborar las proteínas antifúngicas

en estos productos. En este caso, las obtenidas de especies del género Penicillium,

empleadas como cultivos iniciadores, plantearían menos objeciones que las de Aspergillus,

debido a que este último género se relaciona más frecuentemente con la alteración de

alimentos y la producción de micotoxinas (Perrone y col., 2007).

I.3.3. PROTEÍNAS ANTIFÚNGICAS DE Penicillium chrysogenum

En estudios previos se seleccionaron los aislados no toxigénicos de P. chrysogenum

RP42C y AS51D, procedentes de jamón curado, por su capacidad para inhibir el desarrollo

de mohos toxigénicos frecuentes en alimentos. Adicionalmente, se purificaron dos

proteínas con actividad antifúngica, PgAFP y PgChP, a partir de RP42C y AS51D,

respectivamente (Acosta y col., 2009a).

PgAFP y PgChP se obtuvieron como consecuencia de una búsqueda exhaustiva de

proteínas antifúngicas con carga neta positiva, debido a que el carácter catiónico de este

tipo de proteínas se asocia con una menor toxicidad para las células de mamíferos. Así,

PgAFP y PgChP fueron obtenidas mediante cromatografía líquida para la purificación

rápida de proteínas (FPLC) empleando inicialmente columnas de intercambio catiónico y

posteriormente de filtración en gel (Acosta y col., 2009a).

La proteína PgAFP mostró un amplio espectro de inhibición frente a mohos

toxigénicos aislados de jamón curado, tales como A. flavus, A. niger, A. parasiticus, P.

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griseolfulvum, P. solitum o P. restrictum. En cambio, la proteína PgChP sólo afectó a P.

commune, aunque no se descarta que tenga actividad antifúngica frente a otros mohos no

estudiados (Acosta y col., 2009a, b).

Las proteínas PgAFP y PgChP mostraron un peso molecular muy diferente por

SDS-PAGE, siendo de aproximadamente 9 y 37 kDa, respectivamente.

El punto isoeléctrico de PgAFP fue de 9,2, por lo que la proteína resultó ser muy

catiónica. Por otra parte, el hecho de que PgAFP perdiera actividad frente a algunas cepas

del género Aspergillus tras el tratamiento con α-amilasa, sugirió la posibilidad de algún

tipo de glicosilación en la proteína, hecho que se confirmó mediante una tinción de ácido

periódico-Schiff (PAS; Zacharius y col., 1969) siguiendo el método descrito por Deepak y

col. (2003). Además, la secuencia amino terminal parecía indicar que se trataba de una

proteína antifúngica no descrita hasta el momento. No obstante, el análisis mediante

MALDI-TOF de los fragmentos obtenidos de PgAFP tras la digestión con tripsina no

ofreció demasiada información sobre su composición aminoacídica, por lo que apenas se

obtuvieron datos relativos a la estructura primaria de esta proteína (Acosta y col., 2009b).

También se confirmó la producción de PgAFP en condiciones similares a las de las

etapas iniciales de la maduración. Esto podría suponer para P. chrysogenum RP42C una

ventaja adicional para competir frente al resto de mohos que se desarrollan en la superficie

de los productos cárnicos madurados, lo que sería de gran valor para su utilización como

cultivo protector. Además, PgAFP mostró sensibilidad al tratamiento con enzimas como

tripsina, pepsina y lisozima, lo cual permite ser optimistas respecto a su uso en alimentos,

ya que puede ser degradada en el tracto gastrointestinal (Acosta, 2006).

Por otra parte, aunque la información disponible de PgChP se limita a su peso

molecular y a su capacidad para inhibir a P. commune, dadas las diferencias en la masa y

en el espectro de inhibición entre PgAFP y PgChP, en este trabajo se aborda la

caracterización de ambas proteínas.

I.4. CARACTERIZACIÓN DE PROTEÍNAS

Para el estudio de las proteínas se pueden determinar distintas características, entre

las que destacan el peso molecular, el punto isoeléctrico, la secuencia de aminoácidos y la

del gen que la codifica, así como la sensibilidad a distintos tratamientos (térmicos, pHs,

iones, etc.).

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I.4.1. DETERMINACIÓN DEL PESO MOLECULAR

I.4.1.1. Electroforesis en gel de poliacrilamida

Uno de los métodos más habituales para estimar el peso molecular de una proteína

es la electroforesis en gel de poliacrilamida, que puede utilizarse para separar proteínas en

función de su carga y tamaño en condiciones no desnaturalizantes o nativas (Bonnerjea y

col., 1986), o bien sólo en función del tamaño de sus subunidades, en condiciones

desnaturalizantes (Weber y Osborn, 1969). Muchos autores han utilizado la electroforesis

desnaturalizante tanto para comprobar la pureza como para calcular el peso molecular de

proteínas antifúngicas (Lacadena y col., 1995; Marx y col., 1995; Martínez-Ruiz y col.,

1997; Lee y col., 1999).

La electroforesis en gel de poliacrilamida tiene la ventaja de que puede llevarse a

cabo en cualquier laboratorio ya que la inversión en material e instrumentos es mínima. Sin

embargo, este método puede llevar a errores en el peso molecular estimado. De hecho,

proteínas con modificaciones post-traduccionales presentan una movilidad anómala ya que

estas modificaciones provocan que no se una adecuadamente el SDS en electroforesis

desnaturalizantes (Russ y Poláková, 1973; Bagger y col., 2007). Igualmente, proteínas con

puntos isoeléctricos extremos pueden repeler las cargas negativas del SDS y no migrar

como lo deberían hacer teóricamente en función de su peso molecular (Mondstadt y

Holldorf, 1990).

I.4.1.2. Espectrometría de masas

En los últimos años se ha aplicado una técnica muy conocida en otros campos,

aunque novedosa en el estudio de proteínas: la espectrometría de masas. La mayor parte de

los análisis bioquímicos por espectrometría de masas utilizan la ionización por electrospray

(ESI) o por desorción con láser asistida por matriz (MALDI), junto a un analizador de

tiempo de vuelo (TOF) (Ashcroft, 2005). ESI y MALDI son métodos de ionización

“suave” que producen moléculas cargadas con poca fragmentación, aún tratándose de

muestras de muy alta masa molecular.

Tanto en ESI como en MALDI se protona la muestra a analizar y como resultado se

genera un espectro masa/carga (m/z) que tiene una amplia distribución de múltiples iones

cargados. Este espectro m/z puede ser convertido a un espectro con las masas reales

(Figura I3). Manualmente, la masa real puede ser calculada con la fórmula m/z= (M +

nH+)/n, donde M es la masa molecular de la proteína y n es el número de protones

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asociados a ella. El error asociado a esta técnica suele rondar el 0,01% de la masa

(Ashcroft, 2005).

Tanto MALDI como ESI son técnicas ampliamente utilizadas para la determinación

del peso molecular de las proteínas, aunque requieren una fuerte inversión en instrumentos,

así como tener amplios conocimientos en su manejo y en la interpretación de los

resultados. A efectos prácticos, la ionización MALDI suele ser más atractiva porque tiene

mayor tolerancia a sustancias químicas o sales presentes en la muestra, reduciendo así la

necesidad de purificación que requiere la ionización ESI (Standing, 2003). Por lo demás,

ambas son técnicas muy valiosas y precisas para determinar el peso molecular de una

proteína.

I.4.2. DETERMINACIÓN DEL PUNTO ISOELÉCTRICO

Las técnicas más frecuentes para el cálculo del punto isoeléctrico de una proteína

suelen basarse en el isoelectroenfoque, que puede llevarse a cabo mediante electroforesis

en gel en una o dos dimensiones, así como mediante electroforesis capilar.

Figura I3. Determinación del peso molecular mediante espectrometría de masas con

ionización por ESI y MALDI.

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I.4.2.1. Isoelectroenfoque

El isoelectroenfoque es un tipo de electroforesis que permite separar los

componentes de una muestra en función de su punto isoeléctrico. Se basa en la capacidad

de las moléculas de variar su carga neta en función del pH del medio. La electroforesis se

lleva a cabo añadiendo una mezcla de anfolitos con diferentes puntos isoeléctricos en el

medio, para crear un gradiente de pH entre dos electrodos con carga eléctrica opuesta. Al

aplicar una diferencia de potencial cada anfolito se mueve hacia uno u otro electrodo en

función de su propia carga y se detiene cuando el pH del medio que le rodea es igual a su

punto isoeléctrico. Esta técnica se caracteriza por una gran repetibilidad y resolución

(Fuentes Arderiu y col., 1998).

I.4.2.2. Electroforesis bidimensional

Este tipo de electroforesis consiste en realizar dos separaciones electroforéticas

consecutivas de una misma muestra en función de propiedades diferentes en dos

direcciones distintas. Lo más frecuente es realizar la primera separación por

isoelectroenfoque según el punto isoeléctrico, y la segunda en gel de poliacrilamida con

SDS (PAGE-SDS) según la masa molar (Figura I4). Esta técnica se suele emplear para la

separación de muestras complejas de proteínas, pudiendo calcular puntos isoeléctricos,

masas molares, cuantificar proteínas, etc.

Figura I4. Electroforesis bidimensional.

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I.4.2.3. Electroforesis capilar por isoelectroenfoque

La electroforesis capilar es una técnica desarrollada en los últimos años que

consiste en realizar técnicas electroforéticas convencionales (electroforesis en gel,

isoelectroenfoque, etc.) en capilares de sílice fundida con un diámetro interno de entre 25 y

75 µm y una longitud que puede oscilar entre 7 y 100 cm. Tanto uno de los extremos del

capilar como los electrodos se encuentran en una disolución tampón. La muestra se añade

cerca del extremo anódico y se aplica una elevada diferencia de potencial al sistema, que

hace que los componentes de la muestra se muevan a lo largo del capilar y pasen a través

de un detector colocado cerca del cátodo (Holland y Sepaniak, 1993; Berg y col., 2008).

Estudios comparativos entre isoelectroenfoque, electroforesis bidimensional y

electroforesis capilar revelan que las tres técnicas proporcionan una información fiable

acerca del punto isoeléctrico (Valenzuela y col., 1999). Sin embargo, la electroforesis

capilar aporta ventajas como la necesidad de una mínima cantidad de muestra, alta

reproducibilidad y rapidez en los análisis comparativos. A pesar de esto, la electroforesis

bidimensional es una de las técnicas más utilizadas en laboratorios de proteómica para la

separación de mezclas de proteínas y la determinación del punto isoeléctrico.

I.4.3. IDENTIFICACIÓN DE PROTEÍNAS

En la actualidad, en la identificación de proteínas se utilizan diversos

procedimientos, entre los que se incluyen la secuenciación del extremo N-terminal,

detección con anticuerpos específicos, composición de aminoácidos, etc. Todos estos

métodos generalmente son lentos, laboriosos o caros (Gil, 2004).

Hasta hace poco tiempo, para obtener la secuencia aminoacídica de una proteína la

técnica más utilizada era la degradación de Edman. Pero la espectrometría de masas,

debido a su rapidez y elevada sensibilidad, se ha convertido en el método de elección para

la identificación de proteínas a gran escala, que es el primer paso para el estudio de

proteínas de distintos organismos (Russeth y col., 2006; Samyn y col., 2006). Además, esta

técnica también permite la caracterización de modificaciones post-traduccionales que

presentan relevancia fisiológica, tales como la glicosilación y la fosforilación (Claverol y

col., 2003; Morelle y col., 2006). El análisis de las proteínas mediante espectrometría de

masas ha sido posible gracias al desarrollo de varios métodos de ionización suave como

ESI o MALDI para convertir biomoléculas grandes, polares y no volátiles, en iones en fase

gaseosa.

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Esta técnica implica varios pasos: la conversión de los péptidos en fase gaseosa

mediante técnicas de ionización suave como MALDI o ESI; la separación de los iones

según su m/z en un analizador de masas (por ejemplo un analizador de tiempo de vuelo); la

fragmentación opcional de los iones seleccionados, por ejemplo mediante una disociación

inducida por colisión (CID) en un espectrómetro de masas en tándem, combinando dos

analizadores diferentes; y por último la medida de las masas en un detector obteniendo un

espectro de masas que refleja la abundancia de los iones frente a su valor m/z.

Para la identificación de proteínas se han desarrollado dos estrategias que se

encuentran representadas esquemáticamente en la Figura I5: identificación mediante

mapeo peptídico o huella peptídica (PMF: peptide mass fingerprinting) e identificación

mediante etiqueta de secuencia o peptídica, que conlleva la fragmentación de péptidos y la

obtención de la secuencia aminoacídica total o parcial utilizando un espectrómetro de

masas en tándem.

I.4.3.1. Mapeo peptídico o huella peptídica.

El mapeo peptídico o huella peptídica es una técnica utilizada rutinariamente para

identificar proteínas de forma rápida, normalmente a partir de geles de SDS-PAGE o 2D-

PAGE, que se realiza en un espectrómetro de masas tipo MALDI-TOF o ESI-TOF. La

huella peptídica de una determinada proteína es un conjunto de péptidos generados

mediante la digestión con una proteasa específica. Estas masas peptídicas experimentales

son comparadas con las masas peptídicas teóricas de proteínas presentes en bases de datos.

Para ello se han desarrollado diversos algoritmos (PEPSEA, PROFOUND, MS-FIT,

PEPTIDENT, etc) disponibles en Internet. Para la identificación correcta de la proteína se

requiere que las masas de un gran número de péptidos coincidan con sus masas teóricas, y

que cubran parte de la secuencia de la proteína de la base de datos. Además, si la cantidad

de proteína es pequeña, el número de péptidos observados puede ser reducido y por tanto la

proteína no se puede identificar con fiabilidad.

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I.4.3.2. Secuencia peptídica o etiqueta peptídica.

La secuencia peptídica es una estrategia para la identificación de proteínas no

incluidas en las bases de datos o que presentan identificaciones ambiguas mediante

MALDI/ESI-TOF.

Los espectrómetros de masas en tándem MS/MS permiten también la determinación

de la secuencia de aminoácidos. Para ello se selecciona un ión por la masa en un primer

espectrómetro y se fragmenta por colisión con un gas. Posteriormente los fragmentos

obtenidos se analizan en un segundo espectrómetro. Puede utilizarse con una fuente de

ionización tipo MALDI o ESI. A partir de la protonación de los péptidos, estos iones

pueden ser seleccionados y sometidos a una disociación inducida por colisión (CID), en la

Figura I5. Identificación de proteínas mediante espectrometría de masas.

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cual interaccionan con el gas de colisión (normalmente nitrógeno o argón), y tiene lugar la

fragmentación a lo largo del esqueleto peptídico. Los espectros de fragmentación MS/MS

proporcionan información sobre la identidad y posición del aminoácido en el péptido. Por

tanto es posible adquirir la secuencia completa del péptido si se parte de una fragmentación

de buena calidad.

No obstante a veces sólo se obtiene una secuenciación parcial del péptido. La

información sobre la secuencia parcial, la masa del péptido, y la localización exacta de los

aminoácidos, pueden permitir la identificación de la proteína en las bases de datos. A esta

aproximación se le denomina identificación por etiqueta peptídica. Existen diversas

herramientas bioinformáticas que se pueden utilizar para la interpretación de la

fragmentación de un péptido (PEPSEA, SEQUEST, PEPFRAG, MSTAG y MASCOT).

El método de la secuenciación de novo es la estrategia de elección para aquellas

proteínas de las que se desconozca la secuencia del gen que las codifica. El objetivo de esta

aproximación es obtener secuencias completas de aminoácidos que se deduzcan de novo de

los espectros MS/MS (bien mediante interpretación manual o con ayuda de programas

informáticos) y buscar homologías con proteínas presentes en bases de datos. Esta

estrategia permite encontrar proteínas homólogas en otras especies utilizando programas de

búsqueda como BLAST o FASTA.

Finalmente, aunque la proteína de interés pueda no ser identificada o sea una

proteína desconocida, la información sobre las secuencias obtenidas se puede utilizar para

diseñar cebadores que permitan la obtención y clonación del gen que la codifica.

I.5. CARACTERIZACIÓN DEL GEN QUE CODIFICA UNA PROTEÍNA

Caracterizar el gen que codifica una proteína es de gran utilidad porque permite en

muchos casos completar la secuencia de aminoácidos. Adicionalmente, el conocimiento de

la secuencia genética que codifica una proteína permite su clonación en un vector de

expresión y una síntesis más eficiente de la proteína en un organismo distinto del que

normalmente la produce.

I.5.1. BIBLIOTECAS DE ADN COMPLEMENTARIO

La obtención de bibliotecas de ADN complementario o ADNc es una de las

estrategias que se han utilizado con proteínas antifúngicas (Marx y col., 1995). Para ello es

necesario elegir un vector o plásmido que se utilizará como herramienta para la

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construcción de la biblioteca. Los vectores plasmídicos son capaces de clonar fragmentos

de ADNc desde cientos hasta unas 9000 pares de bases, ya que tamaños mayores necesitan

otros vectores como los cósmidos. La elección del plásmido depende de los sitios de

clonación, de restricción y de los lugares de resistencia a antibióticos. Una vez elegido el

vector se une al ADNc sintetizado. Posteriormente se introducen en el microorganismo

competente elegido, formándose los transformantes que constituirán la biblioteca. Los

clones relacionados con la síntesis de la proteína se detectan mediante una sonda de ADN

(Redkar y col., 1996) que puede elaborarse a partir de la secuencia del extremo amino

terminal de la proteína purificada.

Otras estrategias encaminadas a encontrar los genes relacionados con la síntesis de

la proteína están basadas en la realización de PCRs directamente sobre el ADN genómico

(Geisen, 2000) o el ARN mensajero (RT-PCR) del microorganismo productor de la

proteína. Para ello se pueden utilizar cebadores diseñados a partir de las secuencias amino

y carboxilo terminal de la proteína purificada.

I.5.2. AMPLIFICACIÓN RÁPIDA DE EXTREMOS DE ADNc (RACE-PCR)

Existen técnicas que permiten la amplificación de un gen partiendo de un fragmento

conocido de la proteína sin que ésta corresponda necesariamente a los extremos amino o

carboxilo terminal. Una de estas técnicas se basa en la amplificación de extremos de un

gen y es denominada amplificación rápida de extremos de ADNc mediante PCR o RACE-

PCR (“ Rapid Amplification of cDNA Ends - Polymerase Chain Reaction” ).

Para llevar a cabo esta técnica se requiere conocer una parte del gen que queremos

caracterizar. Esta región se puede obtener mediante el diseño de cebadores a partir de

secuencias de aminoácidos obtenidas previamente y la posterior amplificación por PCR

utilizando ADN genómico del microorganismo.

Existen dos tipos de RACE-PCR en función del extremo que se vaya a amplificar:

5’-RACE y 3’-RACE (Scotto-Lavino y col., 2006a y b). En la Figura I6 se representa un

esquema general de la amplificación de un gen completo mediante RACE-PCR. En primer

lugar es necesaria la extracción de ARN a partir de un cultivo del microorganismo en el

cual se ha inducido la transcripción del gen que codifica la proteína. Seguidamente, se

realiza una transcripción reversa empleando unos adaptadores o secuencias conocidas que

hibridan con los extremos 5’ y 3’ del ARN inicial (Figura I7). Por lo tanto, el ADNc

resultante llevará consigo estas secuencias conocidas, generando dos poblaciones de ADNc

INTRODUCCIÓN

19

5’ y 3’ para las amplificaciones de los extremos 5’ y 3’, respectivamente. Para la

amplificación final de los extremos del gen a caracterizar se llevan a cabo dos reacciones

de PCR, una para el extremo 5’ y otra para el extremo 3’, que emplearán respectivamente

las dos poblaciones de ADNc 5’ y 3’ obtenidas anteriormente por retrotranscripción. Se

utilizan además dos cebadores por cada reacción: uno complementario a la secuencia

conocida del adaptador que se unió a los extremos 5’ o 3’ (LUP o SUP), y otro diseñado a

partir de la parte conocida del gen que codifica la proteína (5’-RACE GSP o 3’-RACE

GSP). Para su confirmación, los fragmentos son clonados y secuenciados. Una vez

conocidos los extremos 5’ y 3’, la obtención del gen completo es una simple reacción de

PCR empleando dos cebadores diseñados a partir de los extremos 5’ y 3’ conocidos del

gen.

La determinación de la secuencia de ADN que codifica la proteína antifúngica

puede ser de gran interés para la transformación de otros microorganismos, con el fin de

expresar la proteína con una alta eficacia para su producción a nivel industrial. Esto

facilitaría su utilización como aditivo en alimentos en los que la presencia de mohos no sea

deseable. Por otra parte, también podría servir para expresar la proteína en otros

microorganismos aislados de productos cárnicos madurados que presenten unas

características deseables, pero que sean incapaces de competir con el resto de la población

fúngica.

INTRODUCCIÓN

20

Figura I6. Amplificación de un gen mediante RACE-PCR.

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INTRODUCCIÓN

21

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INTRODUCCIÓN

22

I.6. EVALUACIÓN DE TOXICIDAD EN PROTEÍNAS ANTIFÚNGICAS

Un importante prerrequisito para cualquier aplicación de proteínas antifúngicas es

confirmar su inocuidad mediante ensayos de toxicidad. En este tipo de ensayos se usan

células, tejidos vivos, líneas celulares u organismos, para evaluar los efectos de cualquier

sustancia. Básicamente consisten en su exposición a determinadas concentraciones de la

proteína a evaluar por un tiempo determinado.

El material biológico empleado para los ensayos, ya sean células u organismos,

debe tener alta sensibilidad a los tóxicos, ya que al establecer las concentraciones seguras

para este tipo de material, se espera proteger a los organismos superiores sobre los que se

pretenden emplear las proteínas antifúngicas.

Uno de los métodos comúnmente empleados para demostrar la seguridad y

selectividad de una proteína es el ensayo de actividad hemolítica (Hickey y col., 2003; Jin

y col., 2009). En este método, la evaluación de la posible toxicidad de la proteína objeto de

estudio se realiza empleando glóbulos rojos procedentes de mamíferos y comprobando la

hemólisis producida. Si se produce hemólisis, la proteína tiene efectos citotóxicos y no es

apta para su utilización; si por el contrario la proteína no ocasiona la lisis de los glóbulos

rojos, merecerá seguir siendo estudiada. La ausencia de actividad hemolítica generalmente

se acepta como prueba de que la proteína es segura (Van’t Hof y col., 2001). Algunas

ventajas de la evaluación de la actividad hemolítica con respecto a otros métodos son que

se evita el sacrificio de animales de experimentación y es un método que no requiere

equipos o instrumentos específicos y puede ser realizado en cualquier laboratorio de

investigación. Sin embargo, los ensayos in vitro a menudo no reproducen las condiciones

fisiológicas en los consumidores, donde la proteína puede sufrir cambios que afecten a su

toxicidad (Theis y Stahl, 2004). Por lo tanto, conviene complementar esta prueba con otro

tipo de evaluaciones.

Un método adicional para la comprobación de efectos citotóxicos en proteínas son

los ensayos con líneas celulares, procedentes de células humanas que han perdido la

capacidad de controlar su división celular. La ventaja de las líneas celulares es que tienen

características similares a las células del mismo tipo en el organismo vivo, y a su vez

evitan el sacrificio de animales de experimentación para la obtención de cultivos primarios.

No obstante, ya que las líneas celulares han sufrido ciertas modificaciones en factores

como los del control de la división celular, los cultivos primarios normalmente procedentes

INTRODUCCIÓN

23

de ratas o ratones de laboratorio siguen siendo ampliamente utilizados para la evaluación

de la toxicidad (Szappanos y col., 2005, 2006).

Entre los ensayos que emplean organismos vivos para la evaluación de la toxicidad,

uno de los más útiles y sencillos de desarrollar es el empleo de larvas de Artemia salina.

En este ensayo, las larvas son expuestas a la sustancia a evaluar y se calcula el porcentaje

de mortalidad de las mismas tras un tiempo determinado. La principal ventaja de este

método es que las larvas son especialmente susceptibles a sustancias tóxicas, junto con la

fácil disponibilidad de este organismo. Además, las larvas de Artemia salina no requieren

condiciones especiales de cultivo, simplemente necesitan ser incubadas en un medio que

simula al agua de mar, donde normalmente se desarrolla este organismo. Por todo ello, este

método ha sido ampliamente empleado en múltiples estudios, incluyendo la evaluación de

la presencia de micotoxinas en alimentos (Núñez y col., 1996, 2007; López-Díaz y col.,

2001; González y col., 2007), producción de micotoxinas de hongos patógenos de plantas

(Moretti y col., 2007), en hierbas medicinales (González y col., 2007) o la evaluación de

sustancias empleadas en odontología (Milhem y col., 2008) e incluso de nueva síntesis

(Chohan, 2009).

I.7. EFECTO DE DIFERENTES COMPUESTOS SOBRE LA ACTIVIDAD DE

PROTEÍNAS ANTIFÚNGICAS.

El efecto de diversos tratamientos o sustancias sobre la actividad antifúngica tiene

gran interés para la caracterización de estas proteínas. En primer lugar permitirá formular

hipótesis con respecto a los posibles mecanismos de acción implicados en la actividad que

muestran frente a distintos mohos sensibles. Por otra parte, la influencia de determinados

compuestos o procesos que puedan estar presentes o incorporarse en los alimentos

condicionará en gran medida sus posibles aplicaciones. Por lo tanto, para su

caracterización, se puede estudiar el efecto que ejercen en las proteínas diversos factores

como tratamientos térmicos (Munimbazi y Bulleman, 1998; von der Weid y col., 2003;

Skouri-Gargouri y Gargouri, 2008), diferentes valores de pH (Harighi y col., 2007), o

distintas concentraciones de cationes (Almeida y col., 2000; Kaiserer y col., 2003),

quelantes (Edlind y col., 2002), detergentes (Naeem y col., 2006) o fosfato (Liu y col.,

2002).

INTRODUCCIÓN

24

I.7.1. TEMPERATURA

En diversos alimentos es frecuente la aplicación de tratamientos térmicos, por lo

que la estabilidad de la proteína antifúngica al calor será imprescindible para su posible

aplicación en este tipo de productos.

La termoestabilidad de una proteína está directamente relacionada con su

estructura. De hecho las proteínas resistentes a altas temperaturas poseen gran cantidad de

aminoácidos cargados y residuos aromáticos en su estructura primaria, son frecuentes las

interacciones iónicas o modificaciones post-traduccionales en las mismas, y además son

también muy estables al tratamiento con proteasas (Trivedi y col., 2006; Zhou y col.,

2007). Además, las proteínas plegadas o con gran cantidad de puentes disulfuro son más

resistentes a altas temperaturas.

Las proteínas antifúngicas para uso en alimentos no requieren una alta

termoestabilidad, ya que no irán destinadas a productos sometidos a un tratamiento de

esterilización en los que generalmente no van a sobrevivir microorganismos. Por el

contrario, sí puede ser de interés que resistan tratamientos equivalentes a la pasterización

que se pueden aplicar en productos lácteos o cárnicos.

I.7.2. pH

La evaluación de la estabilidad de las proteínas antifúngicas ante distintos valores

de pH permitiria evaluar su aplicación en alimentos ácidos o fermentados como embutidos

o quesos madurados y encurtidos, siendo el pH de estos últimos incluso menor de 3,7. Del

mismo modo que la temperatura, la resistencia de una proteína a diferentes pHs puede

indicar el grado de estabilidad de su estructura.

I.7.3. CATIONES

El estudio de la influencia de distintos cationes sobre las proteínas es de gran valor

para su caracterización, ya que muchas proteínas antimicrobianas, especialmente las que

actúan sobre membranas, son sensibles a distintas concentraciones de los mismos (Theis y

Stahl, 2004). No obstante existen proteínas antifúngicas, como algunas defensinas de

plantas, que mantienen su actividad incluso con altas concentraciones de iones en el medio

(Thevissen y col., 1999). La presencia de cationes puede además modificar la estructura o

conformación de la proteína (Parker y col., 1996; Sissi y Palumbo, 2009) e incluso

interferir en la unión con las membranas sobre las que actúan. En este tipo de proteínas los

INTRODUCCIÓN

25

cationes pueden interferir en la unión tanto con los fosfolípidos (Osborn y col., 1995;

Thevissen y col., 1997, 1999), como con los receptores específicos sobre los que actúan en

las membranas de los microorganismos sensibles (Almeida y col., 2000; Kaiserer y col.,

2003; Theis y col., 2003). Además en algunos casos, el efecto sobre la actividad de la

proteína es diferente si están presentes cationes monovalentes o divalentes (Broekaert y

col., 1995). Por ello, la exposición de proteínas a diferentes cationes como Ca2+, Mg2+, Na+

o K+, puede ofrecer una valiosa información sobre el tipo de mecanismo de acción

implicado en la actividad antimicrobiana.

Por otro lado, la resistencia a cationes es esencial para la aplicación de proteínas

antimicrobianas en alimentos con alta concentración de sales, como es el caso de algunos

productos cárnicos madurados, ya que estas sales podrían provocar una disminución de la

actividad de la proteína e incluso llegar a inactivarla (Theis y Stahl, 2004).

De manera similar, una baja concentración de iones en el medio también puede

modificar la actividad de una proteína, tanto potenciándola como inhibiéndola (Edlind y

col., 2002). Por ello es necesario evaluar también el efecto sobre la proteína de

secuestradores de iones como EDTA y EGTA. Algunos como EDTA y fosfato suelen ser

empleados como quelantes de metales en alimentos no ácidos que requieren aditivos sin

sabor (Calvo, 1991), por lo que es necesario estudiar la influencia de estos compuestos en

la actividad antimicrobiana si se pretende aplicar la proteína en este tipo de productos.

I.7.4. DETERGENTES

Los detergentes, dependiendo de su carácter iónico, pueden influir en las proteínas

de distinta manera, modificando su estructura e incluso desnaturalizándolas, o potenciando

su actividad al favorecer la solubilidad de sus sustratos.

Los detergentes no aniónicos, como tween 20, tween 80 y Triton X-100, se utilizan

como suplementos en medios de cultivo para favorecer la dispersión de los

microorganismos inoculados. Este tipo de detergentes solubilizan los sustratos sin alterar la

estructura de las proteínas, por lo que no suelen ser muy agresivos contra microorganismos

o proteínas.

Por el contrario los detergentes iónicos, como docecil sulfato sódico (SDS) o ácido

deoxicólico, tienen gran actividad antimicrobiana, ya que ocasionan la disrupción de las

membranas y tienen efectos líticos, por lo que son conocidos como potentes

INTRODUCCIÓN

26

desnaturalizantes de proteínas. Usualmente, estos detergentes son utilizados para el estudio

de proteínas ya que pueden alterar la conformación de las mismas. Así, la adición de

ciertos detergentes puede llevar a la inducción de una estructura en α–hélice en ciertas

proteínas (Naeem y col., 2006).

El uso de detergentes no iónicos como emulsionantes también está extendido en la

industria alimentaria. De hecho en España se emplean tween 20 y tween 80 como

emulsionantes en confitería, repostería y para la elaboración de galletas (Calvo, 1991). La

resistencia de la actividad antimicrobiana de las proteínas a estos detergentes haría que

fuesen unas buenas candidatas para su uso en este tipo de alimentos.

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RESULTADOS

33

Characterization of PgAFP, a novel antifungal protein from Penicillium

chrysogenum

Andrea Rodríguez-Martín, Susan Liddella, Raquel Acosta, Félix Núñez, Miguel A.

Asensio*

Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la

Universidad, s/n. 10071- Cáceres, Spain. a Division of Animal Sciences, University of Nottingham. Sutton Bonington Campus. Loughborough,

Leicestershire. LE12 5RD. United Kingdom.

* Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110.

E-mail: [email protected]

ABSTRACT

A new antifungal protein PgAFP from Penicillium chrysogenum is described. The protein

showed a molecular weight of 6.5 kDa by electrospray ionization mass spectrometry (ESI-

MS). Peptide sequences were obtained by digesting the protein with trypsin and

chymotrypsin and analyzing the resulting peptides by ESI-MS/MS. The obtained

sequences showed high similarity with the antifungal protein Anafp from Aspergillus

niger. No evidence for N- or O- deglycosylations was observed upon chemical and

enzymatic treatments of PgAFP. PgAFP is a new protein that belongs to a group of small,

cysteine-rich, and basic proteins with antifungal activity. However, the characterization of

PgAFP should be exhaustive to demonstrate that this protein is harmless to humans before

any potential use as preservative in foods.

KEY WORDS: antifungal protein, dry-fermented food, growth inhibition, post-

translational modifications.

INTRODUCTION

The antifungal protein PgAFP was purified in previous studies by fast protein liquid

chromatography (Acosta et al., 2009a) from the strain RP42C, which was isolated from

dry-cured ham and characterized as P. chrysogenum based on classical taxonomical

methods (Pitt and Hocking, 1997). PgAFP showed a strong antifungal activity against

selected toxigenic moulds (Acosta et al., 2009a).

MANUSCRITO 1

34

Many fungal proteins have been reported including ribosome inactivating proteins,

antifungal proteins, ribonucleases, ubiquitin-like proteins, lectins, cellulases, xylanases,

laccases, invertases, and trehalose phosphorylases (Ng, 2004). There is only scant

information available about antifungal proteins of fungal origin. PgAFP showed some

similarity (Acosta et al., 2009b) to a group of antifungal peptides secreted filamentous

fungi of the group Ascomycetes, composed of proteins with common characteristics

including being cysteine rich, highly basic, and of low molecular weight. Only four

proteins have been reported in this group: PAF from Penicillium chrysogenum (Marx et

al., 1995), AFP from Aspergillus giganteus (Nakaya et al., 1990; Lacadena et al., 1995),

Anafp from Aspergillus niger (Lee et al., 1999) and AcAFP from Aspergillus clavatus

(Skouri-Gargouri and Gargouri, 2008). The amino acid sequence for all these proteins is

known, except for AcAFP.

In previous studies (Acosta et al., 2009b) PgAFP was positive for glycosylation by

Periodic Acid Schiff reaction (Deepak et al., 2003). No glycosylations have been described

for PAF, AFP, Anafp, and AcAFP, but this kind of post-translational modification has

been described in the antifungal protein from Urginea indica (Deepak et al., 2003) or

chitosanases from Mycobacter AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton

and Eveleigh, 1981).

The aim of this work was to determine the amino acid sequence and molecular weight of

PgAFP. In addition, PgAFP was exposed to enzymatic and chemical deglycosylation

treatments, and analyzed for possible changes in the protein by SDS-PAGE and

electrospray ionization mass spectrometry. The genetic identity of the producer mould was

also confirmed.

MATERIALS AND METHODS

Microbial strain

The strain RP42C was isolated from dry-cured ham, and it was identified as Penicillium

chrysogenum according to classical morphological and biochemical studies (Acosta et al.,

2009a). This mould produces the antifungal protein PgAFP (Acosta et al., 2009a).

RESULTADOS

35

Genetic identification of the producer mould

Genetic characterization of RP42C was carried out by amplifying the fragments 18S-28S

rRNA intergenic spacer (ITS) by polymerase chain reaction (White et al., 1990) and

determining the DNA sequence.

DNA isolation. The mould RP42C was grown in malt extract broth made of glucose 2%

(w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5, at 25ºC for 5 days. After

freezing by adding liquid nitrogen, two grams of mycelium were ground up using a mortar

and pestle. Lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M

Tris-HCl, pH 8, 0.005 M ethylenediamine tetraacetic acid (EDTA), 0.05 M NaCl (TES)

buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Afterwards, 200 µl proteinase K (20

µg/µl) were added, and the mixture was incubated at 60ºC for 35 min, then cooled on ice

and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was

centrifuged at 3,000 ×g for 2 min at 4ºC. The upper phase was transferred to a fresh tube

and DNA was precipitated by the addition of 3 M sodium acetate, pH 5.2 to a final

concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the

pellet was cleaned with ethanol at 70%, centrifuged again, resuspended in sterile water and

treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted with

phenol-chloroform-isoamyl alcohol and precipitated again as indicated above, resuspended

in water and stored at -70ºC. The quantity and quality of the purified DNA was determined

using a Biophotometer (Eppendorf AG, Hamburg, Germany).

ITS amplification. DNA fragments for sequence analysis were obtained by PCR using

primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATAT

GC). Amplified products comprised the internal transcribed spacer 1 (ITS1), 5.8S, and

ITS2 regions of the ribosomal DNA. For each 50 µl reaction, a mixture was prepared

containing 50 ng of genomic DNA, 0.1 vol. of 10× PCR buffer (10 mM Tris-HCl, pH 8.8,

50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, 1 mM of deoxynucleoside triphosphates

(Roche, Mannheim, Germany), 0.4 µM of each primer, and 2 U of DyNAzyme I DNA

Polymerase (Finnzymes, Espoo, Finland). Reactions were run on a programmable thermal

cycler Mastercycler epgradient (Eppendorf AG) with an initial denaturation of 3 min at

96°C, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, with a final

extension at 72°C for 10 min. Aliquots of the PCR were electrophoresed on 1% (w/v)

agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8) using a Sub-Cell

GT apparatus (Bio-rad Laboratories, Hercules, USA). Products were visualized using the

MANUSCRITO 1

36

UV transillumination G-Box (Syngene, Fredericks, USA) and photographed with a

GeneSnap camera (Syngene). DNA fragments were gel-purified and sequenced at the

Institute of Biomedicine (CSIC, Valencia, Spain). The sequences obtained were searched

against the public databases using the Basic Local Alignment Search Tool (BLAST).

PgAFP purification

P. chrysogenum RP42C was grown in malt extract broth at 25ºC for 15 days. The medium

was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and PgAFP

was purified using an ÄKTA fast protein liquid chromatography system (Amersham

Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009a). For this,

filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham

Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound

proteins were eluted with a gradient of NaCl to 1 M (0 to 25% in 15 column volumes

(CV), increasing to 100% in 2 CV, followed by 15 CV at 100%) in 0.02 M sodium acetate

buffer. Eluted proteins were detected at 214 nm. The fraction that showed antifungal

activity in previous studies (Acosta et al., 2009a) was further purified using a HiLoad

26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia Biotech AB)

equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7, at a constant flow

rate of 2.5 ml/min. Data collection and processing were performed using the UNICORN

software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction containing PgAFP

was desalted and concentrated with YM-3 Microcon Centrifugal Filter Units (Millipore

Iberica S.A.U., Madrid, Spain), before storage at -20ºC.

Protein characterization

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE

was performed as described Laemmli (1970). The protein PgAFP was separated on 4%

(v/v) polyacrylamide stacking and 12% (v/v) polyacrylamide resolving gels. Gels were

stained with Imperial Protein Stain (Pierce Biotechnology, Rockford, USA). Two

molecular weight marker sets were used: Precision Plus Protein Standards with ten proteins

between 10 and 250 kDa (Bio-Rad) and Low Molecular Weight Electrophoresis calibration

kit with proteins between 14.4 and 97 kDa (Pharmacia, Uppsala, Sweden).

Molecular weight determination by ESI-MS. The molecular weight of PgAFP was

determined by electrospray mass spectrometry in a Q-TOF2 mass spectrometer (Waters

RESULTADOS

37

Ltd, Manchester, UK). The protein was concentrated and dissolved in ammonium acetate.

PgAFP (0.1 µg/µl) with 1% (v/v) formic acid was desalted using C18 reverse phase pipette

Zip-Tips (Millipore) according to the manufacturer’s instructions. Samples were eluted

into 50% (v/v) methanol, 0.1% (v/v) formic acid, and were loaded into nanospray needles

(Waters) for analysis. The mass spectrometer was operated with a capillary voltage of 900-

1200 V and the sampling cone at 45-50 V. Data were acquired between 400 and 3000 m/z

with a cycle time of 1 second. Mass spectra resulted from combining typically 5 min of

data. Spectra were deconvoluted using the MaxEnt1 maximum entropy software (Waters)

with an output range of 4,000 and 11,000 Da, at a resolution of 1 Da and a peak width of

0.75 Da.

Reduction and alkylation of PgAFP. Reduction and alkylation of the protein prior to

deglycosylation or enzymatic digestion were performed with the protein both in

acrylamide gel pieces and in solution. After SDS-PAGE, PgAFP (25 µg per well) was

stained using Imperial Protein Stain. The band was excised from the gel and washed three

times by incubation in 100 µl of de-stain solution made of 50 mM ammonium bicarbonate

and 50% acetonitrile for 15 min at 40ºC with intermittent vortexing. Gel pieces were

incubated at 56ºC in 100 µl of reducing solution (10 mM dithiothreitol in 50 mM

ammonium bicarbonate) that was removed after 45 min. One hundred µl of alkylation

solution (55 mM iodoacetamide in 50 mM ammonium bicarbonate) was added and

incubation continued at room temperature in the dark for 45 min. The gel slice was then

washed in 100 µl of 50 mM ammonium bicarbonate for 5 min at 40ºC and the supernatant

was discarded. Gel pieces were washed twice in 100 µl of 50 mM ammonium bicarbonate

in 50% (v/v) acetonitrile for 1 min, and centrifuged. Sample was dehydrated in 100 µl of

acetonitrile and incubated for 1-2 min. Finally, the supernatant was discarded and the gel

pieces were dried at 40ºC for a few min. After addition of the digestive enzyme, the tube

containing the gel pieces was cooled at 4°C for 20 min and finally incubated at the optimal

temperature of each enzyme.

To digest PgAFP in solution, different quantities of the protein (12.5, 25 and 62.5 µg) were

added to tubes with 50 mM ammonium bicarbonate up to a volume of 5 µl. Dithiothreitol 5

mM (dissolved in 50 mM ammonium bicarbonate) was added (5 µl) and the final solution

was incubated at 50ºC for 30 min. After adding 10 µl of 20 mM iodoacetamide (dissolved

in 50 mM ammonium bicarbonate), sample was incubated at room temperature for 30 min

in the dark before the digestion with each deglycosidase or protease.

MANUSCRITO 1

38

Analysis for glucidic residues. Chemical deglycosylation was carried out with PgAFP in

solution using two different chemical methods: acid hydrolysis with HCl (Krapf et al.,

2006; Leis et al., 1997) and �-elimination with NaOH (Geert and Thomas-Oates, 1998;

Tarelli, 2007). For acid hydrolysis with HCl, 1 µg of PgAFP was incubated in 20 mM HCl

at 50ºC for 3 h. �-elimination with NaOH was performed incubating 1µg of PgAFP with

0.2 M NaOH at 45ºC for 16 h. Finally, both samples were analyzed by ESI-MS.

Enzymatic deglycosylation was carried out using endoglycosidase F1 (Northstar

BioProducts, Liverpool, UK), PNGase F (New England Biolabs, Ipswich, UK), and the E-

DEGLY commercial kit (Sigma, Madrid, Spain). PgAFP protein (200 µg) was treated with

4 µl of endoglycosidase F1 in 250 mM NaH2PO4 at pH 4.5, the sample was incubated at

37ºC for 1 h, and analyzed by ESI-MS. PNGase F was used following the manufacturer’s

instructions. Two additional assays were performed without detergent with PgAFP in-gel

and in-solution, one with a prior reduction and alkylation as above and another one heating

PgAFP at 100ºC for 5 minutes before adding the enzyme. All samples were analyzed by

SDS-PAGE and ESI-MS. The E-DEGLY deglycosylation kit (Sigma), containing PNGase

F, �-2(3,6,8,9) neuraminidase, O-glycosidase, (1-4)-galactosidase, and �−N-

acetylglucosaminidase, was also used. Reactions were carried out under native and

denaturing conditions according to the manufacturer’s instructions and analysed by SDS-

PAGE.

Amino acid sequencing

Digestion with enzymes

Trypsin digestion. PgAFP was digested with trypsin gold (Promega, Hampshire, UK).

Different treatments with and without reduction and alkylation steps were performed prior

to digestion. Gel piece samples were incubated with trypsin gold (10 ng/µl) in 50 mM

ammonium bicarbonate at 40ºC or 60ºC for 1.5, 3, and 16 h. PgAFP in-solution was

incubated at 37ºC for 16 h in 50 mM ammonium bicarbonate, 5% (v/v) acetonitrile and 10

ng/µl of trypsin gold.

Chymotrypsin digestion. Digestions with the reduced and alkylated protein were performed

with PgAFP in-gel and in-solution. Gel pieces containing PgAFP were digested at different

concentrations (1, 5, 10, and 15 ng/µl) of chymotrypsin (Roche, Welwyn Garden City,

UK) in buffer (100 mM Tris-HCl, 10 mM CaCl2, pH 7.8) at 25ºC and 30ºC for 16 h.

RESULTADOS

39

PgAFP in-solution was digested by chymotrypsin at 0.1, 1, 5 or 10 ng/µl with 5%

acetonitrile and the same buffers detailed above. Temperatures and incubation time were

identical to the in-gel samples.

Electrospray ionization mass spectrometry (ESI-MSMS) analysis for peptide sequence

determination

Peptide samples were made to 0.1% (v/v) formic acid, then desalted using Zip-Tip C18

reverse phase pipette tips (Millipore) according to manufacturer’s instructions. Peptides

were eluted with 50% (v/v) acetonitrile, 0.1% (v/v) formic acid. Samples were loaded into

individual borosilicate nanospray needles (Waters) using 1-10 µl GELoader pipette tips

(Eppendorf) and analyzed in a Q-TOF2 mass spectrometer (Waters), a high resolution

hybrid orthogonal angle-Time of Flight tandem LC mass spectrometer, fitted with a

nanoflow ESI (electrospray ionization) source (Waters). The mass spectrometer was

operated with a capillary voltage of 900–1,200 V in positive ion mode, using argon as the

collision gas. Survey scans of peptide mass spectra were performed with the sampling cone

set at 45–50 V and data were acquired from 400–1,600 m/z with a cycle time of 2.4

seconds. Peptide mass spectra presented resulted from combining typically 5 min of data

acquisition. Individual peptide ions were selected manually for tandem MS analysis.

Collision voltages were optimised manually for individual peptides. Tandem MS

fragmentation spectra were collected typically from 50 to 1,600 m/z. Tandem MS spectra

were deconvoluted into singly charged, mono-isotopic masses using the MaxENT3

maximum entropy software, and peptide sequences were determined by manual

interpretation using the PepSeq software within the MassLynx V 4.0 package (Waters). De

novo peptide sequences were searched against the public database BLASTP using

parameter settings for “short and nearly exact matches”.

RESULTS

Molecular genetic characterisation of P. chrysogenum RP42C

Amplification of the 18S-28S rRNA intergenic spacer (ITS) sequence produced a band

with 394 bp. The DNA sequence of the amplified was determined and compared against

other sequences using BLAST. The sequence from RP42C mould shared 100% of identity

and showed a maximum score with the 18S-28S rRNA intergenic spacer of Penicillium

MANUSCRITO 1

40

chrysogenum strain L1 (Accession number in GenBank: FJ467371). Thus, the producer

mould RP42C was confirmed as Penicillium chrysogenum.

Molecular weight determination

The molecular mass of purified PgAFP was estimated from its migration on SDS-PAGE.

The protein migrated as a single band of approximately 10 kDa (Figure 1).

In ESI-MS, PgAFP resolved as a short series of multiply charged peaks, with the dominant

peak set of around 1,083 Da corresponding to the +6 charge state (Figure 2). The m/z

spectrum was deconvoluted and converted to the molecular mass profile using Maximum

Entropy processing (Figure 3). The mass profile shows a dominant peak of 6,494 Da,

corresponding to the mass of mature PgAFP.

Figure 1. SDS-PAGE of PgAFP (5 µg). M: Pharmacia Low Molecular Weight Markers.

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RESULTADOS

41

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Figure 2. Electrospray mass spectra of PgAFP. A) Spectrum showing the major multiply

charged peaks. B) Expanded spectrum showing the +6 charge state peak set.

MANUSCRITO 1

42

Analysis for glucidic residues

Analysis of PgAFP for glycosylations did not show any evidence of N- or O-linked-

oligosaccharides. Chemical deglycosylation with acid hydrolysis or �-elimination revealed

no changes in the mass spectrum profiles obtained in ESI-MS compared with untreated

controls. No differences on gel-mobility of PgAFP were observed on SDS-PAGE after

attempting deglycosylation by enzymatic means using endoglycosidase F1 (data not

shown), PNGase F (Figure 4), or the E-DEGLY deglycosylation kit (Figure 4). ESI-MS

analysis showed no change in the mass spectrum profiles of PgAFP treated with

endoglycosidase F1 and PNGase F (data not shown).

Figure 3. Molecular mass profile from Maximum Entropy processing. PgAFP resolves as a

single major peak with a mass of 6,494 Da.

6494.29

6592.22

mass

RESULTADOS

43

Peptide sequences

The peptides obtained from PgAFP after digestion by trypsin and chymotrypsin were

analyzed by electrospray ionization tandem mass spectrometry and the amino acid

sequences deduced. Mass spectrum profiles of peptides obtained from PgAFP digestions

are shown in Figure 5. There were no major differences in the peptide profiles for in-gel

compared to in-solution digestions. Six de novo peptide sequences were obtained by

treatment with trypsin and chymotrypsin (Figure 5, Table 1). The survey spectrum and the

processed tandem MS spectrum of a representative peptide are shown in Figure 6.

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Figure 4. SDS-PAGE analysis of attempted enzymatic deglycosylation of PgAFP.

A) PNGase F-treated PgAFP (1) untreated PgAFP (2). B) E-DEGLY deglycosylation kit

under denaturing conditions (1, 2) or native conditions (3, 4). 1 and 3 are untreated controls.

M: Protein molecular weight standards.

MANUSCRITO 1

44

Table 1. De novo obtained peptide sequences. Lower case letters indicate amino acids which were

tentative assignments. Dashes indicate the presence of additional amino acids which were

unassigned. Note that L and I are interchangeable here since these two residues are isobaric.

Sequence

Number de novo sequence

Peptide

Mass

Charge

state

Digestive

enzyme

1 LSKFGGECSL 549.26 +2 Chymotrypsin

2 FGGECSLK 449.24 +2 Trypsin

3 HNTCTYLK 490.26 +2 Trypsin

4a NHVVNCGSAANKK 466.88 +3 Trypsin

4b NHVVNCGSAANK 607.308 +2 Trypsin

5 -yDEHHK 468.19 +2 Trypsin

6 -cqTPV 540.92 +3 Chymotrypsin

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Figure 5. Mass spectra of PgAFP peptides after trypsin (A) and chymotrypsin (B) digestions.

RESULTADOS

45

Figure 6. ESI MS (A) and MS/MS spectrum (B) of a PgAFP tryptic peptide with a charge state

of +3 and precursor mass of 607.308 (peptide 4b NHVVNCGSAANK in Table 1).

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MANUSCRITO 1

46

DISCUSSION

To confirm the previous identification of PgAFP based on morphology and biochemical

characteristics (Acosta et al., 2009a), the 18S-28S rRNA intergenic spacer was analyzed.

The obtained sequence confirmed that the producer mould RP42C belongs to P.

chrysogenum.

The molecular mass of PgAFP obtained by SDS-PAGE was estimated to be over 10 kDa

(Figure 1), whereas ESI-MS revealed a lower mass of 6.5 kDa. For large molecules, such

as intact proteins, ESI-MS is capable of determining the molecular weight to within 0.01%

of the total molecular weight. For a mass of 6,494 Da, this is 0.65 Da. Therefore, it seems

likely that PgAFP migrates anomalously in SDS-PAGE. Many characteristics of proteins

can cause them to migrate atypically on SDS-PAGE, in particular, lipoproteins and

glycoproteins migrate anomalously because SDS cannot bind with lipids or

oligosaccharides (Grefrath and Reynolds, 1974). In previous studies (Acosta et al., 2009b)

PgAFP showed evidences of glycosylations by Periodic Acid Schiff reaction (Deepak et

al., 2003). Of all protein postranslational modifications currently known, such as

acetylation, phosphorylation, formylation, or glycosylation, the last is by far the most

common. Moreover, glycosylated proteins are not uncommon among antifungal proteins,

such as an antifungal glycoprotein from Urginea indica (Deepak et al., 2003) or

chitosanases from Mycobacter AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton

and Eveleigh, 1981). We therefore used a range of enzymatic and chemical

deglycosylation methods to assess possible N-linked and O-linked glycosylation of

PgAFP. We found no evidence of N- or O-linked oligosaccharides. Although in some

cases the assay products were assessed using only SDS-PAGE, in which the resolution

may have been insufficient to reveal relatively small mass changes, no evidence of mass

changes were found using highly resolving MS analysis. Atypical migration on SDS-

PAGE can be due to other causes that do not involve post-translational modifications. For

example, proteins with extreme isoelectric points can show a higher apparent molecular

weight (around a 25% higher) due to electrostatic repulsions causing less SDS to bind and

a consequent decrease in their migration velocity (Mondstadt and Holldorf, 1990).

Notably, the 42 residues obtained using de novo sequencing that together constitute around

72% of PgAFP (a calculated molecular weight of 4.7 kDa, compared with the 6.5 kDa

mass observed by ESI-MS), have a predicted pI of pH 8.8. In addition, in previous studies

RESULTADOS

47

this protein showed an experimental isoelectric point of pH 9.2 by capillary isoelectric

focusing electrophoresis (Acosta et al. 2009b).

Amino acid sequences of PgAFP peptides were obtained using tandem MS and de novo

sequencing (Table 1). The sequences were searched against the public sequence databases

to find any similar proteins present. A multiple alignment was performed with the most

similar proteins (Figure 7).

The PgAFP amino acid sequences had the highest homology with Anafp (81%), and a

hypothetical protein (79 %), both from Aspergillus niger. Three other proteins had lower

levels of similarity with PgAFP; PAF (33%), AFP (26%), and a hypothetical protein from

Aspergillus clavatus (21%). Both PgAFP and PAF are produced by P. chrysogenum, but

they are clearly different proteins. The aligned proteins and PgAFP have common

characteristics: they are small (5.8-6.6 kDa), cysteine-rich, and basic, due to the presence

of a high amount of lysine residues (Marx, 2004). The mature proteins PAF and AFP are

cleavage products of precursor forms which carry a pre-pro-sequence, with a signal peptide

and a prosequence that keep the proteins in an inactive form until exported to the

extracellular medium (Marx, 2004). Since PgAFP may belong to this group of proteins, it

could also be produced as a precursor form. In contrast with other antifungal proteins, such

as some chitosanases, no glycosylations have been described in this group. The apparent

lack of glycosylation of PgAFP is consistent with it being a member of this group.

PgAFP LSKFGGECSLKHNTCTYL Anafp ------------------------------------------LSKYGGECSLEHNTCTYR A.niger ---MQLTSIAIILFAAMGAIANPIAAE---ADNLVARE--AELSKYGGECSVEHNTCTYL PAF ---MQITTVALFLFAAMGGVATPIES---VSNDLDARAEAGVLAKYTGKCTKSKNECKYK AFP MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK A.clavatus MKVVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK PgAFP -K HVVNCGSAANKK yDEHHK cqTPV Anafp -KDGKNHVVSCPSAANLRCKTDRHHCRYDDHHKTVDCQTPV A.niger -KGGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV PAF NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--- AFP AQSGKTAICKCYV---KKCPRDGAKCEFDSYKGKCYC---- A.clavatus AQSGKTAICKCYV---KVCPRDGAKCEFDSYKGKCYC----

Figure 7. Alignment of PgAFP amino acid sequences with the most similar proteins: Anafp

(Lee et al., 1999), XP_001391221 (hypothetical protein from Aspergillus niger CBS 513.88),

AAA92718 (PAF from P. chrysogenum), P17737 (AFP from A. giganteus), and XP_001267787

(hypothetical protein from A. clavatus).

MANUSCRITO 1

48

Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den

Berg et al., 2008). A putative protein sequence is present (accession number CAP80456)

which shows homology with the amino acid sequences obtained from PgAFP in this work.

However, the production of the protein by this mould has not been reported.

PgAFP is a new antifungal protein obtained from P. chrysogenum. Since PgAFP was

produced by a species generally recognized as safe by the United States Food and Drug

Administration (Marx, 2004) it may be permissible for use as preservative in foods.

However, the characterization of PgAFP should be exhaustive to demonstrate that this

protein is harmless to humans. For this, future objectives will include obtaining the coding

sequence of this novel and potent antifungal protein.

ACKNOWLEDGEMENTS

This work was supported by the Spanish Ministry of Education and Science (AGL2004-

06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were recipient of FPI

grants from the Spanish Ministry of Education and Science.

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Manuscrito 2: Genetic characterization of

the gene encoding the novel antifungal protein PgAFP

from Penicillium chrysogenum

RESULTADOS

51

Genetic characterization of the gene encoding the novel antifungal

protein PgAFP from Penicillium chrysogenum

Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa, Miguel A.

Asensio*


Universidad, s/n. 10071- Cáceres, Spain. a Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de

Cáceres s/n, 06071- Badajoz, Spain



ABSTRACT

The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that

inhibits the growth of some toxigenic moulds. The gene encoding the precursor of this

antifungal protein has been characterized. cDNA was prepared from isolated total RNA by

RT-PCR. The cDNA fragment encoding the pgafp gene was obtained by PCR using

degenerate primers based on two known amino acid sequences of the purified PgAFP.

Full-length cDNA of pgafp was obtained by RACE-PCR. Comparison of nucleotide

sequence from genomic fragment and cDNA of the gene, revealed the presence of a 279 bp

coding region which is interrupted by two introns of 63 and 62 bp in length. The precursor

of the antifungal protein consists of 92 amino acids and appears to be processed to the

mature 58 amino acids PgAFP by two-step process. The deduced amino acid sequence of

the preproprotein revealed 75 % identity to an antifungal protein predicted from the

Aspergillus niger CBS 513.88 genome.

KEY WORDS: preproprotein, protective cultures, dry fermented foods.

INTRODUCTION

The strain RP42C of the mould Penicillium chrysogenum secretes a small protein PgAFP

that has been purified for its ability to inhibit toxigenic moulds isolated from dry-cured

meat products (Acosta et al., 2009a,b). Several amino acid sequences of this protein were

obtained by electrospray ionization mass spectrometry (Rodríguez-Martín et al., 2009)

revealing a high homology with the antifungal protein Anafp (Lee et al., 1999). Anafp

protein belongs to a new group of antimicrobial proteins named small, basic and cysteine-

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rich antifungal proteins. The recent sequencing of the genome of different moulds has

shown several genes that could encode proteins with common characteristics to this group.

However, only four proteins have been reported: Anafp from Aspergillus niger (Lee et al.,

1999), PAF from Penicillium chrysogenum (Marx et al., 1995), AFP from Aspergillus

giganteus (Nakaya et al., 1990; Lacadena et al., 1995), and AcAFP from Aspergillus

clavatus (Skouri-Gargouri and Gargouri, 2008). Only AFP and PAF have been genetically

characterizated. In addition a gene homologous to paf gene has been described from

Penicillium nalgiovense (Geisen, 2000).

The genes encoding PAF and AFP have open reading frames interrupted by two introns

with conserved splice sites. They encode products of 92 and 94 amino acids, respectively,

and these proteins are synthesized as a long precursor with a peptide signal and a

presequence that are removed to obtain mature proteins.

Genetic characterization of the gene encoding PgAFP will allow to clon it in other

microorganisms, to increase PgAFP production and to obtain protective cultures for future

applications in foods, environment or medicine. In addition, the genetic sequence will give

valuable information for a better characterization of the protein.


Microbial strain

P. chrysogenum RP42C was isolated from dry-cured ham, and belongs to the fungal

collection of Food Hygiene, University of Extremadura.

DNA isolation

P. chrysogenum RP42C was grown in malt extract broth made with 2% (w/v) glucose, 2%

(w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking for 5

days. The mycelium was obtained by filtering the culture through Whatman paper nº2.

Two grams of mycelium were broken with mortar and pestle after freezing by adding

liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml

0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl

(TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Then, 200 µl proteinase K (20

µg/µl) were added, the mix was incubated at 60ºC for 35 min, cooled on ice, and extracted

with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at

RESULTADOS

53

3000×g for 2 min at 4 ºC. The upper phase containing DNA was transferred to a fresh tube

and precipitated by adding 3 M sodium acetate pH 5.2 to a final concentration of 10% (v/v)

and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with 70%

(v/v) ethanol, centrifuged again in the conditions above mentioned, resuspended in sterile

water, and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was

extrated with phenol-chloroform-isoamyl alcohol, precipitated again as indicated above,

resuspended in water, and stored at -70ºC.

Quantity and quality of purified DNA was determined spectrophotometrically in a

Biophotometer (Eppendorf AG, Hamburg, Germany).

Genomic DNA amplification

Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50

µl reaction mixtures containing 50 pmol each of forward and reverse primers, 0.5 mM each

of the deoxyribonucleotide triphosphates (dNTPs), 0.1 vol of 10× PCR buffer (22.5 mM

MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium sulfate), 1.8 mM MgCl2 and 2.5

units pfu turbo polymerase (Stratagene, La Jolla, USA). The degenerated primers, pgafp-

DPF1 and pgafp-DPR1A (Table 1), were designed from amino acid sequences previously

obtained by mass spectrometry (Rodríguez-Martín et al., 2009). Three reactions were

done: one with both forward and reverse primers, another only with forward, and another

only with reverse primer.

DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific,

Cramlington, UK). The PCR program consisted of initial denaturation (94ºC for 5 min), 35

Primer names Amino acid sequences and designed primers*

pgafp-DPF1

L K H N T C T

5’-TN AAR CAY AAY ACN TGN AC-3’

pgafp-DPR1A

C G S A A N

3’-CR CCN AGN CGN CGN TT-5’

* R for purine, Y for pirimidine, N for A, C, T, or G.

Table 1. Degenerated primers used to amplify the pgafp gene.

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cycles of denaturation (94ºC for 5 s), annealing (45-57ºC range for 5 s, gradient 12), and

extension (72ºC for 1 s) steps, followed by a final extension at 72 ºC for 4 min. PCR

products were electrophoresed on 2% (w/v) agarose gels with MetaPhor agarose (BMA,

Rockland, ME, USA). Two different markers were used for agarose gels: DNA marker

from Gibco (0.15-12kb bands) (Gibco, GrandIsland, NY, USA) and DNA size standard

from Bio-Rad (0.05-2kb) (Bio-rad Laboratories, Madrid, Spain). PCR products detected

only in the reactions with both reverse and forward primers were gel-purified and cloned

into the pCR2.1-TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10

Chemically Competent E. coli (Invitrogen) were transformed with that vector. To confirm

the insert in the vector, digestions with restriction enzyme EcoRI or PCR with M13

forward and reverse primers (Invitrogen) were carried out. Finally, vectors were sequenced

in the Institute of Biomedicine (CSIC, Valencia, Spain).

RNA isolation

P. chrysogenum RP42C was grown in malt extract broth as indicated for DNA isolation.

Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until

RNA extraction. Mycelium was broken with mortar and pestle after adding liquid nitrogen.

RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen) following the

manufacturer’s instructions. The final extracted RNA was resuspended in 40 µl of RNase-

free deionized water.

Rapid amplification of cDNA ends-Polymerase Chain Reaction (RACE-PCR)

Amplification of 5’ and 3’ ends of pgafp gene was carried out using the SMART RACE

cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye,

France).

cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two

separate populations of cDNA were synthesized according to the amplification kit

instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the

SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR

reactions were performed using the Universal Primer A Mix (UPM), in conjunction with

distinct gene-specific primers.

Gene-specific primers design. A gene-specific primer (GSP) was designed for amplifying

the 3’- end (3’-RACE GSP: 5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’) from

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the sequence previously obtained by PCR with degenerate primers pgafp-DPF1 and pgafp-

DPR1A. Amplification of the 5’- end was carried out designing a gene-specific primer (5’-

RACE GSP: 5’-AACTGGGGTCTGGCAGTCAACCCTC-3’) from the 3’-end also

previously obtained.

Polymerase chain reactions were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as

template in 50 µl reaction mixtures containing 1 mM of each of the dNTPs (Roche,

Madrid, Spain), 0.5 µM of the 5’- or 3’-RACE GSP primers, 5 µl of Universal Primer A

Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1 %

Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR

program for obtaining the 3’-end consisted of 35 cycles of denaturation (94ºC for 30s),

annealing (68ºC for 30s), and extension (72ºC for 3 min).

The PCR program for 5’-end amplification was: 5 cycles of 2 steps (94ºC for 30 s and

72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s, 70ºC for 30 s and 72ºC for 3 min) and

27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s and 72ºC for 3 min). Finally, 5’- and 3’-

RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO vectors, which

were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen) before

purifying and sequencing the plasmid.

Digestions with restriction enzyme Eco RI (Roche) or PCR with M13 forward primer

(Invitrogen) were performed to confirm the insert in the vector.

Full-length cDNA amplification. The full cDNA fragment of the gene pgafp was obtained

by PCR with specific primers (Fwd-PrePro pgafp: 5’-ATGCAGATCACCAGCATTGCC-

3’ and Rev-PrePro/Mature pgafp: 5’-TCAAACTGGGGTCTGGCAGTC-3’) designed

from 5’ and 3’ ends. PCR reaction mixtures at a final volume of 50 µl contained 5µl of 5’-

RACE-Ready cDNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the

dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-

100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program

consisted of 35 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and

extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1-

TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E.

coli (Invitrogen) before purifying and sequencing the plasmid. Digestions with restriction

enzyme Eco RI (Roche) or PCR with M13 forward primer (Invitrogen) were performed to

confirm if the insert was in the vector.

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Amplification of the whole pgafp gene

The gene sequence was amplified by PCR using Fwd-PrePro pgafp and Rev-

PrePro/Mature pgafp primers. PCR mixtures were composed by 100 ng of genomic DNA,

0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10×

PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2,

and 2.5 units pfu turbo polymerase (Stratagene) and deionized water until a final volume of

50µl. The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing

(65ºC for 30 s), and extension (72ºC for 3 min). PCR products were cloned in pCR2.1-

TOPO vector that later was used to transform cells of One Shot TOP10 Chemically

Competent E. coli. The plasmid was purified and sequenced. Introns sequences were

confirmed by GenScan software.

RESULTS

Partial amplification of pgafp gene using degenerated primers

The pgafp gene partial amplification was done using the two degenerate primers, pgafp-

DPF1 and pgafp-DPR2A (Table 1). After different reactions, the optimal annealing

temperature was 45.1ºC. PCR products run in agarose gel showed one small band of

around 75 bp that was amplified only in reactions with both the forward and the reverse

primers (Figure 1). This band was selected for cloning and sequencing.

Figure 1. Products obtained by PCR using DPF1 primer (F), DPF1+DPR2A primers (F+R),

and DPR2A (R) primer. M: DNA standards. The arrow indicates the selected band for

cloning and sequencing.

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RESULTADOS

57

After sequencing, the fragment found was 70 bp length (5’-tgaagcataatacgtgcacatacctaaag

ggtggaaagaaccatgtagtcaattgcggttctgccgctaa-3’), and the translated sequence (KHNTCTYL

KGGKNHVVNCGSAA) was compared in Basic Local Alignment Search Tool (BLAST)

showing common sequences with Anafp from Aspergillus niger.

Sequence of pgafp gene by RACE-PCR

Both 3’ and 5’ -ends of the gene pgafp were obtained by amplification from cDNA. mRNA

was extracted from a 5 days-old culture of P. chrysogenum RP42C and was used to obtain

the two different populations of 5’- and 3’-RACE-Ready cDNA by RT-PCR. The gene

specific primer 3’-RACE GSP (5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’),

designed from the known pgafp sequence of genomic DNA, allowed to amplify the 3’-end

by RACE-PCR. The resulting product of about 450 bp included 121 bp of the 3’-end of

pgafp (Figure 2). Amplification of the 5’-end was carried out with a new gene specific

primer, 5’-RACE GSP (5’-AACTGGGGTCTGGCAGTCAACCCTC-3’), designed from

3’-end sequence. The obtained band, of approximately 440 bp, included the full pgafp

cDNA gene.

Amplifications of complete gene in genomic DNA and cDNA were performed by

designing two new primers Fwd-full pgafp (5’-ATGCAGATCACCAGCATTGCC-3’) and

Rev-full pgafp (5’-TCAAACTGGGGTCTGGCAGTC-3’). The sequence of genomic DNA

was 404 bp, whereas the amplified cDNA was 279 bp (Figure 3). The difference between

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Figure 2. cDNA fragments of 5’- and 3’-RACE PCR (A and B, respectively).

M: DNA standards.

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the sequences revealed the existence of two introns of 63 and 62 bp (Figure 4), absent in

the cDNA, which were confirmed by GenScan software.

The open reading frame was translated to protein by ExPASy proteomics server at the

Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase

Figure 3. pgafp gene amplification of the with genomic DNA (1) and cDNA (2). M: DNA

standards.

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5’-atgcagatcaccagcattgccattgtcttcttcgccgcaatgggtgcggttgctaacccc M Q I T S I A I V F F A A M G A V A N P atcgcgagggagtcggacgatcttgatgcccgagacgtacagcttagtaaattcggagga I A R E S D D L D A R D V Q L S K F G G gtaagttcttcttacaagacgtctatatagaaatagcactaacctttctgaaccacttta << Intron 1 caggaatgcagcttgaaacacaacacgtgcacatacctaaagggtggaaagaaccatgta >> E C S L K H N T C T Y L K G G K N H V gtcaattgcggttcggccgccaacaagaaggtaggttccgattcgattcggggccaattg V N C G S A A N K K << Intron 2 atttgttcttatcatttaatcttcatctacagtgcaagtctgatcgccaccactgtgaat >> C K S D R H H C E acgatgagcaccacaagagggttgactgccagaccccagtttga-3’ Y D E H H K R V D C Q T P V -

Figure 4. Full-length DNA sequence of pgafp gene. The open reading frame has been translated

to amino acids. Consensus splice sequences and internal marker of fungal introns are underlined.

RESULTADOS

59

cleavage site predicted by the program SignalP (Nielsen et al., 1997) suggested that the

pre-sequence consisted of the first 18 amino acids (MQITSIAIVFFAAMGAVA).

DISCUSSION

P. chrysogenum RP42C produces a high amount of the PgAFP in the first week of

incubation (Acosta, 2006). Thus, to obtain appropriate cDNA populations, a 5 days-old

culture was used to make sure that the mRNA from gene encoding PgAFP was actively

being produced. The RACE-PCR was effective to obtain pgafp gene. Thus, the designed

primers and obtained cDNA populations were adequate to amplify the 3’ and 5’ –ends of

pgafp. When the complete sequence was obtained, a difference between the pgafp gene

from genomic DNA (404 bp) and from cDNA (279 bp) could be observed (Figure 3).

Thus, the existence of two introns of 63 and 62 bp in the gene, which are absent in the

cDNA, was confirmed by GenScan software. The small size of these introns compared to

those of mammalian introns is a typical feature of fungal genes (Gurr et al., 1987). The 5’-

and 3’-ends of the two introns are quite similar (Figure 4) and coincide with the consensus

splice sequences for fungal introns: 5’-splice donor site GTNNGT (N= A, G, T or C) and

the consensus 3’-splice acceptor site DYAG (D= A, G or T; Y= C or T). In addition, the

first intron has the internal CTAAC sequence, very common in fungal introns (Wiesner et

al., 1988).

The pgafp open reading frame encodes a 92 amino acid-long precursor of the PgAFP. The

first 18 amino acids correspond with a secretion signal sequence according to the SignalP

program (Nielsen et al., 1997). The next residues (19-34) were not found in the amino acid

sequence obtained from the purified protein (Rodríguez-Martín et al., 2009). These 16

amino acids constitute a prosequence that would be removed before or during the release

of mature PgAFP. Prosequences play an important role by preventing protein activity

before secretion. Only after the prosequence is cleaved off, the mature protein adopts its

correct tertiary structure and gains its final activity. Antifungal proteins produced as

preproproteins, such as PAF and AFP (Nakaya et al., 1990; Marx et al., 1995), have been

described from other moulds.

The deduced mature PgAFP has 58 amino acid residues. It shows a high percentage (27.6

%) of basic amino acids. The isoelectric point was estimated to be 8.8 using the Compute

pI tool from Expasy (Gasteiger et al., 2005). The isoelectric point of PgAFP estimated by

isoelectrofocusing in previous studies (Acosta et al., 2009b) was 9.2, higher than the

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predicted value with Compute pI tool. However, the prediction can be not exact for highly

basic and small proteins (Gasteiger et al., 2005). A pI of 9.2 results in a net positive charge

under physiological conditions, which is a common characteristic among antifungal

proteins from moulds.

The accessibility of the whole genome sequence of an increasing number of

microorganisms facilitates the search for genes encoding potential antifungal proteins.

Three of these genes, those from Aspergillus niger, Neosartorya fischeri, and Gibberella

zeae, show partial homology with PgAFP protein. The sequences of the hypothetical

proteins from these genes as well as other reported antifungal proteins are aligned in Figure

5. The protein showing the highest homology (75%) with the precursor of PgAFP was the

hypothetical protein from A. niger. Despite both PAF and PgAFP precursors are produced

by Penicillium chrysogenum, they only share a similarity of 43%.

There are six cysteines conserved in all the similar proteins (Figure 5). Those residues

would play an important role on stabilizing of the tertiary structure of PgAFP by formation

of disulfide bridges (Nakaya et al. 1990; Marx et al., 1995; Lee et al., 1999).

PgAFP ---MQITSIAIVFFAAMGAVANPIARE---SDDLDARD--VQLSKFGGECSLKHNTCTYL Anafp ------------------------------------------LSKYGGECSLEHNTCTYR A.niger ---MQLTSIAIILFAAMGAIANPIAAE---ADNLVARE--AELSKYGGECSVEHNTCTYL N.fischeri ---MQITKISLFLFVGIGVVASPIHAE---SDGLNARAVNAADLEYKGECFTKDNTCKYK G.zeae ---MQFSTIIPLFVAAMGVVATPVNSP---AQELDARGNLFPRLEYWGKCTKAENRCKYK PAF ---MQITTVALFLFAAMGGVATPIESV---SNDLDARAEAGVLAKYTGKCTKSKNECKYK AFP MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK A.clavatus MKVVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK PgAFP K-GGKNHVVNCGSAANKKCKSDRHHCEYDEHHKRVDCQTPV Anafp K-DGKNHVVSCPSAANLRCKTDRHHCRYDDHHKTVDCQTPV A.niger K-GGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV N.fischeri ID-GKTYLAKCPSAANTKCEKDGNKCTYDSYNRKVKCDFRH G.zeae NDKGKDVLQNCPKFDNKKCTKDGNSCKWDSASKALTCY--- PAF NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--- AFP AQSGKTAICKCYV---KKCPRDGAKCEFDSYKGKCYC---- A.clavatus AQSGKTAICKCYV---KVCPRDGAKCEFDSYKGKCYC----

Figure 5. Alignment of PgAFP amino acid sequences (Rodríguez-Martín et al., 2009) with

other antifungal proteins. Accession numbers in Genbank are XP_001391221 (hypothetical

protein from Aspergillus niger CBS 513.88), XP_001262586 (hypothetical protein from

Neosartorya fischeri NRRL 181), XP_384921 (hypothetical protein from Gibberella zeae PH-

1), AAA92718 (PAF from P. chrysogenum), P17737 (AFP from A. giganteus) and

XP_001267787 (hypothetical protein from Aspergillus clavatus). Common amino acids are

denoted by shaded background letters.

RESULTADOS

61

In previous studies PgAFP showed a molecular weight of 6,494 Da by mass spectrometry

(Rodríguez-Martín et al., 2009). Translating the genetic sequence according to Gasteiger et

al. (2005) the predicted mass of the mature protein was 6,500 Da, very close to the weight

determined by mass spectrometry. In previous works, PgAFP protein was tested for having

glycosylations by treatments with deglycosilases and chemical compounds, and no linked-

oligosaccharides were found. The minimal difference found between predicted and

experimental molecular weights confirms that this protein is not glycosylated.


Berg et al., 2008). A putative protein can be found in BLAST (accession number

CAP80456) that differs from the precursor of PgAFP in only three amino acids. However,

the production of the protein by this mould has not been reported.

In conclusion, the gene enconding the antifungal protein PgAFP has been sequenced. Such

as similar proteins, PgAFP is synthesized as a long precursor with a peptide signal and a

presequence that are removed to obtain mature proteins. The characterization of pgafp

gene will permit its cloning for overproduction or heterologous expression of the protein in

other microorganisms.

ACKNOWLEDGEMENTS


06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of FPI

grants from the Spanish Ministry of Education and Science. Authors would like to thank

Dr. Susan Liddell (Division of Animal Sciences) and Prof. Ian Connerton (Division of

Food Sciences) for their support during Andrea Rodriguez stay’s at Nottingham

University.

REFERENCES

Acosta, R. Doctoral Thesis: “Selección de Penicillium productores de péptidos

antifúngicos para su utilización en productos cárnicos madurados”. University of

Extremadura. Spain. 2006.




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Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D.,

and Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server;

(In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press.

Full text - Copyright Humana Press (2005).

Geisen, R. P. nalgiovense carries a gene which is homologous to the paf gene of P.

chrysogenum which codes for an antifungal peptide. Int. J. Food Microbiol. 62: 95-101

(2000).

Gurr, S.J., Uncles, S.E., and Kinghorn, J.R. The structure and organization of nuclear

genes of filamentous fungi. In: Kinghorn, J.R. (Ed.). Gene structure in Eukaryotic

Microbes. IRL Press, Oxford, pp. 93-193 (1987).


Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., Gavilanes, J.G.

Characterization of the antifungal protein secreted by the mould Aspergillus giganteus.

Arch. Biochem. Biophys. 20: 273-281 (1995).

Lee, D.G., Shin, S.Y., Maeng, C-Y., Jin, Z.Z., Kim, K.L. and Hahm, K-S. Isolation and

characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophy.

Res. Commu. 263: 646-651 (1999).

Marx, F., Haas, H., Reindl, M., Stöffler, G., Lottspeich, F. and Redl, B. Cloning,



(1995).

Nakaya, K., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H., and Ulbrich,

N. Amino acid sequence and disulfide bridges of an antifungal protein isolated from

Aspergillus giganteus. Eur. J. Biochemistry. 193: 31-38 (1990).

Nielsen, H., Engelbrecht, J., Brunak, S., and Von Heijne, G. Identification of

prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein

Eng. 10: 1-6. (1997).

RESULTADOS

63

Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A.

Characterization of PgAFP, a novel antifungal protein from Penicillium chrysogenum.

Manuscrito 1 (2009).

Skouri-Gargouri, H. and Gargouri, A. First isolation of a novel thermostable antifungal





Roubos, J.A., van der Klei, I.J., van Peij,N.N., Veenhuis,M., von Dohren, H., Wagner,

C., Wortman, J. and Bovenberg, R.A. Genome sequencing and analysis of the


Wiesner, P., Beck, J., Beck, K.F., Ripka, S., Müller, G., Lücke, S. and Schweizer, E.

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patulum. Eur. J. Biochem. 177: 69-79 (1988).

Manuscrito 3: Preliminary evaluation of the toxicity and

sensitivity to different treatments of the antifungal protein

PgAFP from Penicillium chrysogenum

RESULTADOS

65

Preliminary evaluation of the toxicity and sensitivity to different

treatments of the antifungal protein PgAFP from Penicillium

chrysogenum

Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Miguel A. Asensio*


Universidad, s/n. 10071- Cáceres, Spain.



ABSTRACT

The protein PgAFP obtained from Penicillium chrysogenum RP42C inhibits toxigenic

moulds found in foods. The aim of this work was to evaluate the potential toxicity of

PgAFP and to study basic characteristics of its antifungal activity, including the effect of

treatments and compounds common in foods. No haemolytic activity or toxicity on

Artemia salina larvae was detected. PgAFP showed neither chitinase nor chitosanase

activities. The protein showed a high stability to heat treatments up to 100ºC for 15 min,

and at pH values from 1 to 12. Some detergents potentially present in food as indirect

additives interfered with the antifungal activity, but tween 20 had no impact on the protein.

Only high concentration of other compounds, such as EDTA and sodium phosphate,

affected PgAFP activity when low levels of the protein were present. Monovalent ions

showed very little impact, whereas Ca2+ blocked the antifungal activity even at low

concentrations. These results indicate that PgAFP could be used to prevent mould growth

in foods with high salt levels or where thermal or acid stability are essential.

KEY WORDS: antifungal activity, food preservative, protective culture.

INTRODUCTION

Penicillium chrysogenum has been proposed as starter culture for mould fermented foods

(Martín et al., 2002; Benito et al., 2005), mainly due to its ability to produce volatile

compounds that contribute to flavour development (Ramírez et al., 1995; Jacobsen and

Hinrichsen, 1997; Martín et al., 2006). However, the continuous evolution of the physico-

chemical characteristics along ripening can favour the development of undesirable moulds,

which would not only lead to a negative flavour of the product (Lund et al., 1995), but also

MANUSCRITO 3

66

to mycotoxin production (Núñez et al., 1996, 2000; Nielsen et al., 1998; López-Díaz et al.,

2001; Sosa et al., 2002; Sabater-Vilar et al., 2003). Some mould can produce different

antifungal compounds, including proteins. The use of natural antifungal proteins could be

attractive to satisfy consumer's demand. Therefore, fungal cultures with antagonistic

activity against spoilage and toxigenic moulds could be of interest to improve the

microbial safety of the product.

In previous studies, P. chrysogenum RP42C isolated from dry-cured ham showed a broad-

spectrum inhibition against toxigenic moulds, including Aspergillus niger, A. flavus, and P.

commune due to the protein PgAFP (Acosta et al., 2009a, b).

Various mechanisms of action have been described for antifungal proteins, including

interference in synthesis of DNA, RNA, or proteins, damage to cellular ribosome (Hwu et

al., 2000), and membrane channel and pore formation (Thevissen et al., 1999).

Nevertheless, the most useful antifungal proteins are those specific against fungal

membrane and cell wall, because of their lesser toxic effects on mammalian cells (Groll et

al., 1998). Cationic antifungal proteins, such as AFP from A. giganteus and PAF from P.

chrysogenum, have proved to be specific for fungal membranes (Marx et al., 2008; Meyer

et al., 2008) and not for mammalian cells (Szappanos et al., 2005 and 2006). Interestingly,

PgAFP shows similar characteristics to AFP and PAF, both in the amino acid sequence and

the isoelectric point (Marx, 2004; Acosta et al., 2009b; Rodríguez-Martín et al., 2009a, b).

A prerequisite for any application of antifungal proteins or the producer mould on foods is

the lack of toxicity for consumers. The use of antifungal proteins from ascomycetes can be

limited by chemical compounds present and physical treatments applied to foods. Many

antifungal proteins, especially membrane-acting peptides, are sensitive to cations, which

can be a crucial point in the application to food products with high salt concentrations

(Theis and Stahl, 2004). Given that PgAFP is produced by a mould isolated from a

substrate with high salt concentration, its antifungal activity could withstand high-cation

levels. Moreover, antifungal proteins should also be active under the singular conditions of

foods where moulds can grow, particularly in a wide range of pH values. Other compounds

that can be present on foods, such as detergents and chelators, may also interfere with

antifungal proteins. In addition, stability to heat treatments can be of utmost interest for the

potential use of antifungal proteins as food preservative.

The aim of this work was to carry out a preliminary evaluation of the potential toxicity of

RESULTADOS

67

PgAFP, as well as the impact of salts, heat treatments, different pH values, and detergents

on its antifungal activity.


Microbial strains

The PgAFP producing mould, P. chrysogenum RP42C, and the toxigenic moulds

Penicillium restrictum Pr341 and Aspergillus niger An261, were isolated from dry-cured

ham and belong to the fungal collection of Food Hygiene (University of Extremadura,

Spain).

PgAFP purification

P. chrysogenum RP42C was grown at 25ºC for 15 days in malt extract broth made of

glucose 2% (w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5. Then, the

medium was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and

PgAFP was purified using an ÄKTA fast protein liquid chromatography system

(Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009a).

For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column

(Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH

4.5. Bound proteins were eluted with the following gradient of 0.02 M sodium acetate, 1 M

NaCl buffer, pH 4.5: 0% to 25% in 15 column volumes (CV), increase to 100% in 2 CV,

and finally keeping 100% for 15 CV. Eluted proteins were detected at 214 nm. The

fraction that showed antifungal activity in previous studies (Acosta et al., 2009a) was

purified using a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham

Pharmacia Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer,

pH 7, at a constant flow rate of 2.5 ml/min. Data collection and processing were done

using the UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the

fraction containing PgAFP was desalted and concentrated with YM-3 microcon centrifugal

filter units (Millipore Iberica S.A.U., Madrid, Spain) and kept at -20ºC.

Protein was quantified by Johnson method according to Córdoba (1990). An aliquot of the

purified protein (100 µl) was dried at 100 ºC. The sample was digested by adding 200 µl of

sulfuric acid at 120 ºC. Then, 4.8 ml of distilled water, 3 ml of 4 N NaOH, and 2 ml of

Nessler reactive made of KI 0.4% (w/v), 0.4% HgI2 (w/v), and 0.175% (w/v) arabic gum

MANUSCRITO 3

68

were added. After 10 min at room temperature, the absorbance was measured at 490 nm in

a spectrophotometer. A calibration curve was obtained with different concentrations of

ammonium sulfate.

Sodium dodecyl sulphate-polyacrylamide gel eletrophoresis (SDS-PAGE)

SDS-PAGE was performed as described Laemmli (1970). The protein PgAFP was

separated on a 4% (v/v) polyacrylamide stacking and 8% (w/v) polyacrylamide resolving

gels. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford,

U.S.A.). The molecular weight was determined by comparison of its electrophoretic

mobility with proteins of the molecular mass marker (6.5-66 kDa) from Sigma (Sigma-

Aldrich Química S.A., Madrid, Spain).

Haemolytic activity assay

The haemolytic activity of the protein was determined using sheep defibrinated blood

(Oxoid). The erythrocytes were isolated by centrifugation (1,000 ×g for 6 min at room

temperature) and washed three times with 50 mM sodium phosphate, 0.15 M NaCl buffer,

pH 7. The cell concentration in the stock suspension was adjusted to 1×109 cells/ml. A

volume of 50 µl of cells suspension was added to 200 µl of sterile concentrated protein

(300 µg/ml) and incubated at 37ºC. After 0, 8, and 24 h, the samples were placed on ice,

immediately centrifuged at 4ºC, and the supernatant was carefully collected. The

haemolysis was determined by measuring the absorbance of the supernatant at 570 nm

(Moerman et al., 2002) and 541 nm (Lai et al., 2005). Controls for 0% and 100%

haemolysis were obtained suspending sheep red blood cells in the buffer or distilled water,

respectively. The haemolysis percentage was calculated using the following equation:

Percentage haemolysis of supernatants = [(Abs in the peptide solution − Abs in buffer)/Abs

in water solution] × 100.

Three independent experiments were conducted with triplicate readings for each one.

Toxicity assay using brine shrimp larvae (Artemia salina)

The toxicity of PgAFP was evaluated using brine shrimp larvae, following the Harwig and

Scott method (1971) with modifications. Brine shrimp medium (BSM) was made of 0.3%

(w/v) dipotassium glycerophosphate, 3% (w/v) NaCl, 0.03% (w/v) CaCl2 • 2H2O, 0.05%

RESULTADOS

69

(w/v) MgSO4 • 7H2O, 0.15% (w/v) MgCl2 • 6H2O, 0.08% (w/v) KCl, 0.01% (w/v)

MgBr2 • 6H2O, and 0.6% (w/v) glycine. The medium was adjusted to pH 6.5. Brine

shrimp larvae (100-200 mg) were grown in 100 ml BSM under continuous shaking (140

rpm) for 30 h at 30ºC. The test was carried out in Erlenmeyer flasks containing 20 to 40

larvae in 10 ml BSM. PgAFP was added at final concentrations of 3, 16, 33, 57, and 75

µg/ml. Buffer without PgAFP was used in negative controls. Dead larvae were counted just

before incubation and after 24 h at 30ºC in a shaker at 80 rpm. Finally, the mortality

percentage was calculated. PgAFP was indicated as nontoxic when death rate of larvae was

not significantly different (p <0.05) from spontaneous mortality in controls.

Evaluation of chitinase and chitosanase activities

Chitinase and chitosanase activities were determined with a modification of the 3,5-

dinitrosalicylic acid method (Su et al., 2006) by measuring the reducing sugars liberated

during the hydrolysis of chitin or chitosan. One ml of the 1% (w/v) chitin or chitosan

(Sigma-Aldrich) in 1% (v/v) acetic acid was added to 1 ml of PgAFP (300 µg/ml) and

incubated for 2-3 h at 37ºC. Then, to detect the hydrolisis reaction, 2 ml of dinitrosalicylic

acid reagent, made of 1% (w/v) 3,5-dinitrosalicylic acid, 0.05% (w/v) sodium sulphite and

1% (w/v) phenol dissolved, were added and the sample was incubated at 100ºC for 45 min.

Finally, 1 ml of 40% (w/v) potassium sodium tartrate was added and the solution was

centrifuged. The supernatant was measured at 520 and 575 nm. To quantify the reducing

sugars liberated a calibration curve was obtained with different concentrations of D-(+)-

Glucosamine hydrochloride (Sigma-Aldrich). A chitinase from Streptomyces griseus

(Sigma-Aldrich) with both chitinase and chitosanase activities was used as positive control.

Preparation of colloidal chitin: Colloidal chitin was obtained following the method of

Roberts and Selitrennikott with minor modifications (Harighi et al., 2007). Ten grams of

chitin powder were added slowly into 100 ml of H3PO4 (85%) at 25ºC under vigorous

shaking for 2 h. This solution was carefully added over 1 liter of absolute ethanol at -20ºC.

The mix was shaken for 30 min and stored until use. Ten ml of suspension were

centrifuged, the pellet was washed three times with 50 mM sodium phosphate buffer pH 7,

and finally resuspended in distilled water. The final concentration of colloidal chitin was

around 1%.

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Chitinase activity after gel electrophoresis

The chitinase activity assay was carried out as described by Trudel and Asselin (1989).

PAGE-SDS was performed in 12% (v/v) polyacrylamide gradient gel containing 0.01%

(w/v) glycol chitin and 0.1% (w/v) SDS. A stacking gel with 4% (v/v) polyacrylamide

containing 0.1% (w/v) SDS was used. Samples were boiled for 5 min with 20% (v/v)

glycerol, 0.025% (w/v) bromophenol blue, 2% (w/v) SDS and 5% (v/v) �-mercaptoethanol

in 62.5 mM Tris-HCl (pH 6.8). The gel was run at 100 V for 1 h.

After electrophoresis, the gel was incubated for 2 h at 37 ºC under continuous shaking in

100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100. Then, the gel

was stained with a 0.01% (w/v) calcofluor white M2R (Sigma-Aldrich) in 500 mM Tris-

HCl (pH 8.9). After 5 min, this solution was removed and the gel was incubated in distilled

water at room temperature for about 1 h. Lytic zones were visualized in the UV

transilluminator Syngene G-Box (Synoptics, Fredericks, USA). The gel was photographed

and analyzed with the GeneSnap software (Synoptics). An additional gel was stained only

with Imperial Protein Stain (Pierce Biotechnology, Rockford, U.S.A.). The chitinase from

S. griseus (Sigma) was used as positive control. The molecular mass marker used was 66-

6.5 kDa (Sigma).

Glycol chitin synthesis: 0.2 g glycol chitosan was dissolved in 4 ml of 10 % (v/v) acetic

acid and the mixture was shaken overnight at 22ºC. Methanol (18 ml) was slowly added

and the solution was filtered through 0.45 µm pore size cellulose acetate membrane filters.

Acetic anhydride (0.3 ml) was added and kept for 30 min. The resulting gel was cut into

small pieces and after adding 1 vol of methanol was shaken for 4 min. This suspension was

centrifuged at 27000 ×g at 4ºC for 15 min and the pellet was resuspended in 20 ml of

0.02% (w/v) sodium azide. The final concentration of the glycol chitin was 1% (w/v).

Evaluation of antifungal activity

Quantitative assay for fungal growth inhibition was performed following the protocol

developed by Broekaert et al. (1990) with some modifications (Acosta et al., 2009a). The

toxigenic strains P. restrictum Pr341 and A. niger An261 (Núñez et al., 1996; Díaz, 1999)

were used as reference strains. To perform the inhibition test in microtitter plates, 100 µl of

the fraction obtained by gel filtration chromatography were dispensed in triplicate wells.

Each well was loaded with 100 µl of concentrated malt extract broth (4% malt extract, 4%

RESULTADOS

71

glucose and 0.2% peptone, pH 4.5) inoculated with the reference strain to c.a. 106

spores/ml. The plates were incubated for up to 96 h at 25ºC. Fungal growth was

determined by measuring the optical density variation at 595 nm after 96 h.

Effect of heat-treatments

The study of the protein stability after heat treatment was carried out according to Okkers

et al. (1999). Aliquots of PgAFP protein (300 µg/ml) were exposed to different

combinations of temperatures (60, 80, and 100ºC) and times (10, 20, and 30 min), as well

as to sterilization at 121ºC for 15 min. After the heat-treatment, the samples were cooled

on ice and tested for antifungal activity in microtiter plates.

PgAFP antifungal activity after exposure at different pH values

PgAFP (300 µg/ml) was retained in microcon centrifugal filter units YM-3 (Millipore) and

then dissolved in different buffers at the following values: pH 1, 2 (HCl/KCl); pH 3

(glycine/HCl); pH 4, 5, 6 (citric acid/sodium phosphate); pH 7 (sodium phosphate/NaCl);

pH 8 (Tris/HCl); pH 9, 10 (Tris/NaOH); and pH 12 (KCl/NaOH). Samples were incubated

at 25ºC for 2 h and the pH adjusted to 4.5 before testing the antifungal activity.

Effect of different chemicals on PgAFP antifungal activity

To study the influence of some chemicals on the antifungal activity, several assays were

carried out with 3, 30, and 300 µg PgAFP/ml in presence of the following compounds:

NaCl, CaCl2, KCl, or MgCl2 (0.1, 1, 10, and 100 mM); EDTA or EGTA at 0.01, 0.1, and 1

mM; 1% of Triton X-100, tween-20, tween-80, SDS, or deoxycholic acid; NaH2PO4 at 0,

0.1, 1, 10, and 100 mM.

Statistical analysis

Data were statistically analyzed by SPSS software package (version 13.0. SPSS Inc.,

Chicago, USA) for a variance analysis (one way ANOVA) and the means were separated

by the Tukey honest significant difference test.

MANUSCRITO 3

72

RESULTS

Evaluation of PgAFP toxicity

None of the readings at either 570 or 541 nm during the incubation time revealed

haemolytic activity. The concentration of PgAFP tested (240 µg/ml) showed 0%

haemolysis after 24 h.

In an additional assay with Artemia salina, the larvae mortality was 0% even at the highest

concentration of the protein 75 µg/ml after 24 h incubation. Therefore, PgAFP showed no

toxicity at any concentration tested.

Chitosanase and chitinase activities

No chitosanase or chitinase activity was observed by the 3,5-dinitrosalicylic acid method,

as no glucosamine was liberated by PgAFP in the conditions assayed.

To further evaluate the chitinase activity, chitin hydrolysis was tested by electrophoresis in

a glycol chitin gel. No activity was observed loading the polyacrylamide gel with 50 µg of

PgAFP (Figure 1). Lytic zones by chitinase from S. griseus used as positive control were

observed as black bands after staining with calcofluor white M2R.

Figure 1. Chitinase activity by gel electrophoresis. SDS-PAGE with glycol chitin was

stained with Imperial Protein Stain (A) and Calcofluor White M2R (B). M: protein marker,

Q: chitinase from S. griseus (100 µg).

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RESULTADOS

73

Effect of heat-treatments and different pH values

PgAFP protein showed a remarkable stability at high temperatures. PgAFP kept antifungal

activity against P. restrictum and A. niger even after being heat-treated at 80ºC for 30 min

or 100ºC for 15 min (Figure 2).

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Figure 2. Antifungal activity of heat-treated PgAFP expressed as mean and standard deviation of

reference moulds growth after 96 h. Asterisks indicate statistically significant differences to

positive control with non heat-treated PgAFP (***p<0.001). C+: positive control with non heat-

treated PgAFP; C-: negative control without PgAFP. a: Autoclave cycle.

Incubation in buffers at pH values from 1 to 12 had no effect on the stability of PgAFP and

the protein remained active against the reference moulds (Figure 3).

MANUSCRITO 3

74

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Figure 3. Antifungal activity of PgAFP after treatment at different pH values expressed as mean

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Effect of cations

The impact of different monovalent and divalent cations at 0.1, 1, 10, and 100 mM on the

inhibitory activity of PgAFP (3, 30, and 300 µg/ml) over P. restrictum and A. niger was

tested (Figure 4). Monovalent cations did not seem to exert a strong impact on the

antifungal activity. The antifungal activity on A niger decreased only with at the highest

concentration of K+ or Na+ (100 mM) and the lowest level of PgAFP. The remaining

conditions tested did not alter the activity of PgAFP against P. restrictum and A. niger.

Divalent cations showed a deeper impact on PgAFP activity against both sensitive moulds.

The highest level (100 mM) of Ca2+ decreased PgAFP activity, irrespectively of the protein

concentration tested. Even 10 mM Ca2+ reduced the inhibition with 3 µg/ml of PgAFP.

Mg2+ showed a lower effect than Ca2+, given that only 100 mM decreased the activity with

low PgAFP concentrations.

RESULTADOS

75

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Figure 4. Effect of monovalent (A) and divalent (B) cations on antifungal activity of PgAFP

expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate

statistically significant differences to the negative control without PgAFP (*p<0.05; **p<0.01;

***p<0.001).

Effect of calcium chelators

EDTA and EGTA showed a different effect on PgAFP activity. EGTA had no influence on

PgAFP antifungal activity, even at the highest concentration tested (1 mM). EDTA at

MANUSCRITO 3

76

levels up to 0.01 mM had no impact on PgAFP antifungal activity against either sensitive

mould, whereas 0.1 mM limited the activity of lower levels of PgAFP just on P. restrictum

(Figure 5). The highest concentration of EDTA (1 mM) prevented growth of P. restrictum

and A. niger (data not shown), and no further effect could be established.

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Figure 5. Effect of EDTA on the antifungal activity of PgAFP expressed as mean and standard

deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant

differences to the negative control without PgAFP (***p<0.001).

Effect of detergents

Even though non-ionic detergents limited the growth of the reference moulds, it was

possible to evaluate their effect on PgAFP activity. However, SDS totally prevented

growth of reference moulds, and no effect on PgAFP could be established. For both

sensitive strains, tween 20 had no effect, tween 80 only reduced the activity with 3 µg/ml

of PgAFP, and Triton X-100 decreased the activity at all PgAFP concentrations (Figure 6).

With deoxycholic acid PgAFP did not show antifungal activity against P. restrictum,

whereas the effect of PgAFP on A. niger could not be observed due to the extremely poor

growth of this mould.

RESULTADOS

77

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Figure 6. Effect of Triton X-100 (T100), tween 20 (T20), tween 80 (T80), and deoxycholic acid

(DAc) on the antifungal activity of PgAFP expressed as mean and standard deviation of reference

moulds growth after 96 h. Asterisks indicate statistically significant differences to the negative

control without PgAFP (**p<0.01; ***p<0.001).

Effect of sodium phosphate

Sodium phosphate influenced the antifungal activity against both reference moulds,

particularly on A. niger (Figure 7). With P. restrictum, the effect was limited to high levels

of phosphate (10-100 mM) with the lowest PgAFP concentration. However, the lowest

phosphate concentration (0.1 mM) produced a significant effect on the activity against A.

niger with 3 µg/ml of PgAFP. This effect increased gradually with the phosphate

concentration.

MANUSCRITO 3

78

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Figure 7. Effect of phosphate on the antifungal activity of PgAFP expressed as mean and standard

deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant

differences to the negative control without PgAFP (**p<0.01; ***p<0.001).

DISCUSSION

P. chrysogenum RP42C has been proposed as protective culture on mould fermented foods

(Acosta et al., 2009b), and the use of PgAFP as additive could also be of interest to prevent

unwanted moulds.

Common methods to evaluate safety are the haemolytic assay (Theis et al, 2004) and the

brine shrimp larvae (Artemia salina) test (Harwig and Scott, 1971; Milhem et al., 2008;

Chohan, 2009). The absence of cytolytic activity to red blood cells is generally accepted as

proof that the protein can be regarded as safe (Van’t et al., 2001). Brine shrimp larvae

(Artemia salina) test has been also used to screen fungal toxins (Harwig and Scott, 1971)

or synthetic compounds (Milhem et al., 2008; Chohan, 2009) for toxicity. The results here

presented revealed neither haemolytic activity against red blood cells nor lethal effects on

brine shrimp larvae when PgAFP was tested up to 240 and 75 µg/ml, respectively.

The information available on other antifungal proteins from moulds reveals that neither

PAF nor AFP was toxic up to 100 µg/ml to human umbilical vein endothelial cells

(Szappanos et al., 2005, 2006). There are also bacterial antifungal proteins non-toxic to

blood red cells, such as PPEBL21 from Escherichia coli at 1,250 µg/ml (Yadav et al.,

2007). On the other hand, some antifungal proteins from bacteria have shown toxic effects.

RESULTADOS

79

SAP protein isolated from Streptomyces sp. strain AP77 was non-toxic up to 250 µg/ml to

human dermal fibroblasts but toxic at all higher doses (Woo et al., 2002). A more potent

toxicity was found for syringomycin, a protein that has shown 100% haemolysis at 20

µg/ml (Sorensen et al., 1996). Antifungal peptides purified from Bacillus cereus were also

reported to be highly toxic to human erythrocytes (Latoud et al., 1986). The toxicity of

most reported antifungal proteins to mammalian cells has become a major limitation for

their use to develop new antifungal drugs or preparing improved antifungal formulations.

Therefore, the lack of toxicity showed by PgAFP in these preliminary tests supports its

potential for food use, although a more complete evaluation is required.

It is known that most chitin containing organisms produce chitinases, which are

presumably required for morphogenesis of cell walls and exoskeleton (Gooday, 1971).

Apart from these functions, these enzymes have also been found to be involved in the host

defence against pathogens (Somashekar and Joseph, 1996). Chitinases from moulds have

been described to have antifungal activity, such as from Thermomyces lanuginosus (Guo et

al., 2005) and from Fusarium chlamydosporum (Mathivanan et al., 1998). In addition, in a

parallel work an antifungal protein from P. chrysogenum AS51D that hydrolyses

deacetylated chitin has been described (Rodríguez-Martín et al., 2009c). However, PgAFP

did not show either chitinase or chitosanase activities when tested in solution or in gel

(Figure 1). Therefore, the antifungal activity of this protein cannot be based on the

degradation of these compounds commonly found in fungal cell wall.

Some detergents can be present on various foods as a consequence of their use as direct or

indirect additives. Detergents are unique among protein denaturants, and some of them can

alter protein conformation at low concentrations. The addition of detergents can either

increase or decrease the helical conformation, and even convert � forms into helices

(Naeem et al., 2006). The effect of ionic and non-ionic detergents on PgAFP varied

according to the sensitive mould and the protein concentration tested (Figure 6). Triton X-

100 and deoxycholic acid inhibited the antifungal activity, irrespectively of PgAFP

concentration. However, the inhibitory effect of both tweens was limited to tween 80 at the

lowest PgAFP concentration tested. Ionic detergents tend to denature proteins by altering

native three dimensional structures. Some ionic detergents, such as deoxycholic acid, can

also act as open-channel blockers, as it has been shown for porins (Duret and Delcour,

2006). No further information is available to suggest whether the effect of deoxycholic

acid is mainly due to changes in the tertiary structure of PgAFP or in the fungal membrane.

MANUSCRITO 3

80

In this study, the non-ionic detergent Triton X-100 seems to counteract the antifungal

activity of PgAFP. However, the partial inhibition on growth of reference moulds made it

difficult to evaluate the particular impact of Triton X-100 on PgAFP activity.

Finally, the activity of PgAFP decreased as sodium phosphate concentration increased,

although the protein remained active up to 100 mM of phosphate.

Among the main factors that can interfere with the antifungal activity in foods are salts, pH

and heat treatments. PgAFP was highly resistant to a wide range of temperature and pH

values. As it is shown in figure 2, PgAFP displayed antifungal activity after treatment at

80ºC for 30 min, and even at 100ºC for 15 min. Similarly, incubation in buffers at pH

values from 1 to 12 had no effect on PgAFP antifungal activity. Additionally, PgAFP

showed a high stability to proteases, such as pepsin and papain (Acosta, 2006). These

characteristics are similar to those of other antifungal proteins from moulds. AFP from A.

giganteus is stable at high temperature, protease degradation, and a broad pH range due to

a specific folding pattern (Lacadena et al., 1995). The mature form of AFP folds into five

antiparallel � strands which define a small and compact �-barrel, stabilized by internal

disulfide bridges formed by eight cysteine residues (Campos-Olivas et al., 1995). It has

been assumed that Anafp, NAF, and PAF have the same tertiary structure, although only

three disulfide bridges can be formed by the six cysteine residues present in these proteins

(Marx, 2004). PgAFP has common characteristics to these antifungal proteins, including a

high stability, six cysteine residues, and a quite high similarity in the amino acid sequence

(Rodríguez-Martín et al., 2009a, b). Thus, PgAFP could also have a tertiary structure based

on a compact �-barrel.

On the other hand, cation sensitivity has been described for basic antimicrobial proteins,

including AFP and PAF. In fact, AFP activity decreases in the presence of Ca2+, K+, and

Na+ ions (Theis et al., 2003), and PAF activity is sensitive to Mg2+, K+ and Na+ (Kaiserer

et al., 2003). Cations seem to disturb the interaction of antifungal proteins with negatively

charged membrane components (Barkai-Golan et al., 1978) or specific receptors

(Broekaert et al., 1992). The latter has been suggested for AFP (Theis et al., 2003) and

PAF (Kaiserer et al., 2003).

The influence of high cation levels on PgAFP depended on the ion charge, protein

concentration and the sensitive mould. The highest effect was obtained with the divalent

cations (Figure 4). Defensins are also more sensitive to divalent than to monovalent cations

RESULTADOS

81

(Terras et al., 1992), particularly to Ca2+ (Lehrer et al., 1989; Cociancich et al., 1993;

Osborn et al., 1995). This indicates that the effect of monovalent cations depended on the

reference mould, with no effect on the activity against P. restrictum. The antagonistic

effect of ions over plant defensins is dependent on the fungus and thus on the conformation

of the putative target site (Terras et al., 1992; Osborn et al., 1995). Thus, the distinct effect

of cations on PgAFP activity against each sensitive mould tested can be explained by

differences on receptor conformation. In addition, the results obtained for PgAFP suggest

that the ratio of protein to ion concentration plays a decisive role on the interaction with

protein activity.

EDTA can also be present in foods due to its use as metal sequestrant additive. The ability

of EDTA and EGTA for chelating ions is well known, being EDTA the most potent of

them (Choucair et al., 2006). However, EDTA chelates both Ca2+ and Mg2+, whereas

EGTA is more specific for Ca2+ ions. EGTA did not influence PgAFP activity, whereas

EDTA inhibited this activity only against P. restrictum at the lower concentrations of the

protein tested (Figure 5). Thus, an excess of divalent cations has a stronger effect on

PgAFP activity than the lack of those ions.

In summary, PgAFP protein is stable over a wide range of pH values and active in

presence of high concentrations of ions and at high temperatures. All this information

revealed that PgAFP has potential to prevent mould growth in a wide range of foods with

high salt levels or where thermal or acid stability are essential. However, the use of this

protein as additive requires an in-depth toxicity evaluation.

ACKNOWLEDGEMENTS


06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of a

FPI grant from the Spanish Ministry of Education and Science.

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Manuscrito 4: Characterization of PgChP, a novel chitosanase

with antifungal activity from Penicillium chrysogenum

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Characterization of PgChP, a novel chitosanase with antifungal activity


Andrea Rodríguez-Martín, Susan Liddella, Raquel Acosta, Félix Núñez, Miguel A.

Asensio*


Universidad, s/n. 10071- Cáceres, Spain. a Division of Animal Sciences, University of Nottingham. Sutton Bonington Campus. Loughborough,

Leicestershire. LE12 5RD. United Kingdom.



ABSTRACT

A new fungal chitosanase with antifungal activity, PgChP from Penicillium chrysogenum,

is described. Two isoforms were found by SDS-PAGE in the purified extract of PgChP.

After enzymatic deglycosylation only the smaller isoform was observed by SDS-PAGE.

Identical amino acid sequences were obtained from the two proteins and these showed a

high similarity with fungal chitosanases. Analysis of the molecular mass by electrospray

ionization mass spectrometry (ESI-MS) revealed the existence of six peaks from 30 to 31

kDa differentiated by a mass multiple of 162 Da, the typical mass of galactose or mannose

residues. This could be due to different levels of glycosylation of the same protein PgChP.

The isoelectric point was aproximately pH 5 estimated by two-dimensional polyacrylamide

gel electrophoresis. PgChP is the first chitosanase to be described from P. chrysogenum.

KEY WORDS: antifungal protein, food preservative, glycosylation, N-linked

oligosaccharides, protective culture, toxigenic mould.

INTRODUCTION

The antifungal protein PgChP was selected in previous studies (Acosta et al., 2009a) from

the strain AS51D isolated from dry-cured ham and characterized as Penicillium

chrysogenum according to classical taxonomical methods (Pitt and Hocking, 1997). PgChP

was purified by fast protein liquid chomatography and showed antifungal activity against

selected toxigenic moulds (Acosta et al., 2009a).

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Antifungal proteins have been described from diverse organisms including plants and

animals, but only scant information is available about antifungal proteins of fungal origin.

Many antifungal proteins have interesting applications because they attack compounds of

fungal walls that are not present in mammalian cells, such as glucanases, chitinases and

chitosanases (Adams, 2004). Fungal infections and contaminations have led to an

increasing demand for antifungal drugs in diverse fields including agriculture, medicine

and food protection. Many antifungal drugs have low efficacy rates, show severe side

effects and cause resistance on the microorganisms, as antibiotics (Meyer, 2008). This

makes it necessary to study new compounds, including fungal proteins such as PgChP.

However, before any application of PgChP, an exhaustive characterization of the protein is

required.

In previous studies, another antifungal protein from P. chrysogenum, named PgAFP,

showed evidence of glycosylation by periodic acid-Schiff staining (Acosta et al., 2009b).

Glycan chains in glycoproteins participate in many biological processes, such as protein

interactions, or confer stability on the protein against proteases or heat treatments (Morelle

et al., 2006). These modifications introduce variations in the molecular mass and net

charge of the protein. Enzymatic and chemical deglycosylation treatments followed by

SDS-PAGE and electrospray ionization mass spectrometry are commonly used to evaluate

this sort of post-translational modification (Claverol et al., 2003). Additional information

on the molecular mass and amino acid sequences is typically obtained by mass

spectrometry after digestion in-gel or in-solution with proteolytic enzymes (Habermann et

al., 2004).

This paper describes the amino acid sequencing, molecular mass determination and

isoelectric point estimation of the antifungal protein PgChP.


Microbial strain

P. chrysogenum AS51D was isolated from dry-cured ham, being identified according to

classical morphological and biochemical studies (Acosta et al., 2009a).

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91

Genetic identification of the mould

Genetic characterization of AS51D was carried out by amplifying the fragments 18S-28S

rRNA intergenic spacer (ITS) by polymerase chain reaction (White et al., 1990).

DNA isolation. The mould AS51D was grown in malt extract broth, made of glucose 2%

(w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5, at 25ºC for 5 days. Two

grams of mycelium were broken with morter and pestle after freezing by adding liquid

nitrogen. Lysis was performed by homogenizing the powder in 4 ml 0.05 M Tris-HCl, pH

8, 0.005 M ethylenediamine tetraacetic acid (EDTA), 0.05 M NaCl (TES) buffer and 1%

(w/v) sodium dodecyl sulfate (SDS). Afterwards, 200 µl proteinase K (20 µg/µl) was

added, and the mixture was incubated at 60ºC for 35 min, cooled on ice and extracted with

phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 3,000 ×g

for 2 min at 4ºC. The upper phase containing DNA was transferred to a fresh tube and

precipitated by the addition of 3 M sodium acetate pH 5.2 to a final concentration of 10%

(w/v) and two volumes of cold ethanol. After centrifugation, the pellet was washed with

70% (v/v) ethanol, centrifuged again in the conditions mentioned above, resuspended in

sterile water and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA

was extracted with phenol-chloroform-isoamyl alcohol and precipitated again as indicated

above, resuspended in water and stored at -70ºC. The quantity and quality of purified DNA

was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg,

Germany).

ITS amplification. PCR fragments for sequence analysis were obtained using primers ITS1

(TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) covering

the internal transcribed spacer 1 (ITS1) region, 5.8S, and ITS2 region of the ribosomal

DNA. For each 50 µl reaction, a mixture was prepared containing 50 ng of genomic DNA,

0.1 vol. of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4

mM MgCl2, 1 mM of deoxynucleoside triphosphates (Roche, Mannheim, Germany), 0.4

µM of each primer, and 2 U of DyNAzyme I DNA Polymerase (Finnzymes, Espoo,

Finland). Reactions were run on a programmable thermal cycler Mastercycler epgradient

(Eppendorf AG) with an initial denaturation of 3 min at 96°C, followed by 30 cycles at

94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 10 min.

Aliquots of the PCR were electrophoresed on 2% (w/v) agarose gels in 1× TAE buffer (40

mM Tris-acetate, 1 mM EDTA) pH 8, using a Sub-Cell GT apparatus (Bio-rad

Laboratories, Hercules, USA). Products were visualized using the UV transillumination G-

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Box (Syngene, Fredericks, USA) and photographed with a GeneSnap camera (Syngene).

DNA fragments were gel-purified and sequenced at the Institute of Biomedicine (CSIC,

Valencia, Spain). The sequences obtained were searched against the public databases using

the Basic Local Alignment Search Tool (BLAST).

PgChP purification

P. chrysogenum AS51D was grown in malt extract broth at 25ºC for 15 days. The medium

was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and PgChP

was purified using an ÄKTA fast protein liquid chromatography system (Amersham

Bioscience AB, Uppsala, Sweden) according to Acosta et al. (2009a). For this, filtered

medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham Pharmacia

Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound proteins were

eluted with a gradient of NaCl to 1 M (0 to 25% in 15 column volumes (CV), increasing to

100% in 2 CV, followed by 15 CV at 100%) in 0.02 M sodium acetate buffer. Eluted

proteins were detected at 214 nm. The fraction that showed antifungal activity in previous

studies (Acosta et al., 2009a) was purified using a HiLoad 26/60 Superdex 75 prep grade

gel filtration column (Amersham Pharmacia Biotech AB) equilibrated with 50 mM sodium

phosphate, 0.15 M NaCl buffer pH 7, at a constant flow rate of 2.5 ml/min. Data collection

and processing were done using the UNICORN software 4.12 (Amersham Pharmacia

Biotech AB). Finally, the fraction containing PgChP was desalted and concentrated with

YM-10 Microcon Centrifugal Filter Units (Millipore Iberica, Madrid, Spain) and stored at -

20ºC.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed as described Laemmli (1970). The protein PgChP was

separated on a 4% (v/v) polyacrylamide stacking and 8% (v/v) polyacrylamide resolving

gel. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Rockford, USA).

Two preparations of molecular weight markers were used: Precision Plus Protein

Standards with ten proteins between 10 and 250 kDa (Bio-Rad) and the Low Molecular

Weight Electrophoresis calibration kit with proteins between 14.4 and 97 kDa (Pharmacia,

Uppsala, Sweden).

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93

Reduction and alkylation of PgChP

Reduction and alkylation of the protein prior to deglycosylation or enzymatic digestion

were performed with the protein in SDS-PAGE gel pieces or in solution. After SDS-

PAGE, PgChP (25 µg per well) was stained using Imperial Protein Stain. The band was

excised from the gel and washed three times by incubation in 100 µl of de-stain solution

(50 mM ammonium bicarbonate, 50% acetonitrile) for 15 min at 40ºC with intermittent

vortexing. Gel pieces were incubated at 56ºC in 100 µl of reducing solution (10 mM

dithiothreitol in 50 mM ammonium bicarbonate) which was discarded after 45 min and

replaced with 100 µl of alkylation solution (55 mM iodoacetamide in 50 mM ammonium

bicarbonate) and incubation continued at room temperature in the dark for 45 min. The gel

pieces were then washed in 100 µl of 50 mM ammonium bicarbonate for 5 min at 40ºC

and the supernatant was discarded. The gel was washed twice in 100 µl of 50 mM

ammonium bicarbonate in 50% acetonitrile for 1 min and centrifuged. Sample was

dehydrated in 100 µl of acetonitrile and incubated for 1-2 min. Finally, the supernatant was

discarded and the gel pieces were dried at 40ºC for a few min. After addition of the

digestive enzyme, the tube containing the gel pieces was cooled at 4°C for 20 min and

finally incubated at the optimal temperature of each enzyme.

For digestion of PgChP in-solution, different quantities of the protein (1.25, 2.5, and 6.25

µg) were added into tubes containing 50mM ammonium bicarbonate up to a final volume

of 5 µl. Dithiothreitol 5 mM dissolved in 50 mM ammonium bicarbonate was added (5 µl)

and the final solution was incubated at 50ºC for 30 min. After adding 10 µl of 20 mM

iodoacetamide dissolved in 50 mM ammonium bicarbonate, sample was incubated at room

temperature for 30 min in the dark before digestion with each enzyme.

Digestion with enzymes for amino acid sequencing

Trypsin digestion. Digestion with the reduced and alkylated protein was performed with

PgChP in-gel and in-solution. Gel pieces containing PgChP were incubated with 10 ng/µl

trypsin gold (Promega, Hampshire, United Kingdom) in 50 mM ammonium bicarbonate at

40 or 60ºC for 1.5, 3, or 16 h. Additionally, further automated in-gel digestions were

carried out. For these, samples of PgChP in-gel pieces were de-stained, dehydrated,

reduced, alkylated, washed, and digested using the ProteomeWorks MassPREP robotic

liquid handling station (Waters, Manchester, UK), essentially as described above. For the

digestion step, the microtiter plate containing the gel pieces was cooled to 6°C for 10 min

MANUSCRITO 4

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before addition of 25 µl per well of trypsin gold at 10 ng/µl. The plate was incubated at

6°C for 20 min and incubated at 37°C for 4 h. PgChP was digested in-solution by

incubating at 37ºC for 16 h after adding 50 mM ammonium bicarbonate, containing 5%

acetonitrile and 10 ng/µl of trypsin gold. All digested protein samples were stored at 4°C

until ESI-MS analysis.

Chymotrypsin digestion. Digestion with the reduced and alkylated protein was performed

with PgChP in-gel and in-solution. Gel pieces were digested at different concentrations (1,

5, 10, and 15 ng/µl) of chymotrypsin (Roche, Welwyn Garden City, UK) in buffer (100

mM Tris-HCl, 10 mM CaCl2 buffer pH 7.8), at 25ºC and 30ºC for 16 h. PgChP in-solution

was digested by chymotrypsin at 0.1, 1, 5, or 10 ng/µl with 5% (v/v) acetonitrile and the

same buffer mentioned above. Temperatures and incubation time were identical to those

used for in-gel sample digestions.


Chemical deglycosylation was carried out with PgChP in solution and using two different

chemical methods: acid hydrolysis with HCl (Leis et al., 1997; Krapf et al., 2006) and �-

elimination with NaOH (Geert and Thomas-Oates, 1998; Tarelli, 2007). For acid

hydrolysis with HCl, 1 µg of PgChP was incubated in 20 mM HCl at 50ºC for 3 h. �-

elimination with NaOH was performed by incubating 1µg of PgChP in 0.2 M NaOH at

45ºC for 16 h. Finally, both samples were analyzed by ESI-MS.

Enzymatic deglycosylation

PNGase F (New England Biolabs, Ipswich, UK) was used following the manufacturer’s

instructions and samples were analyzed by SDS-PAGE.

E-DEGLY deglycosylation kit (Sigma, Madrid, Spain) contained the following

deglycosylases: PNGase F, �-2(3,6,8,9) neuraminidase, O-glycosidase, (1-4)-galactosidase,

and �−N-acetylglucosaminidase. Reactions were carried out under native and denaturing

conditions according to manufacturer’s instructions and products were subjected to SDS-

PAGE analysis.

RESULTADOS

95

Electrospray ionization mass spectrometry (ESI-MS) analysis for peptide sequence

determination

After deglycosylation and enzymatic digestion, formic acid was added to a final

concentration of 0.1% (v/v); then the sample was desalted using Zip-Tip C18 reverse phase

pipette tips (Millipore) according to manufacturer’s instructions. Peptides were eluted with

an equal volume of 50% (v/v) acetonitrile and 0.1% (v/v) formic acid. Samples were

loaded into individual borosilicate nanospray needles (Waters) using 1-10 µl GELoader

pipette tips (Eppendorf) and analyzed in a Q-TOF2 mass spectrometer (Waters). ESI-MS

was carried out with a capillary voltage of 900–1200 V in positive ion mode, using argon

as the collision gas. Survey scans of peptide mass spectra were performed with the

sampling cone set at 45–50 V and data were acquired from 400–1500 m/z with a cycle

time of 2.4 seconds. Peptide mass spectra presented resulted from combining typically 5 –

15 min of data acquisition. Individual peptide ions were selected manually for tandem MS

analysis. Collision voltages were optimised manually for individual peptides. Tandem MS

fragmentation spectra were collected typically from 50 to 1600 m/z. Tandem MS spectra

were deconvoluted into singly charged, mono-isotopic masses using the MaxENT3

maximum entropy software and peptide sequences were determined by manual

interpretation using the PepSeq software within the MassLynx V 4.0 package (Waters,

UK). De novo peptide sequences were searched against the public databases using

BLASTP with parameter settings for “short and nearly exact matches". A multiple

sequence alignment of fungal chitosanases was produced using the program T-Coffee at

www.ebi.ac.uk.

Molecular weight determination by ESI-MS

The molecular weight of PgChP was determined by electrospray mass spectrometry in a Q-

TOF2 mass spectrometer (Waters, UK). PgChP (0.125 µg/µl) in 1% (v/v) formic acid was

desalted using C18 reverse phase pipette Zip-Tips (Millipore) according to the

manufacturer’s instructions. Protein was eluted into 50% (v/v) methanol, 1% (v/v) formic

acid, and loaded into nanospray needles for analysis. The mass spectrometer was operated

with a capillary voltage of 900-1200 V and the sampling cone at 45-50 V. Data were

acquired between 400 and 3000 m/z with a cycle time of 1 second. Mass spectra resulted

from combining typically 5 min of data. Spectra were deconvoluted using the MaxEnt1

MANUSCRITO 4

96

maximum entropy software with an output range of 20,000 and 40,000 Da, at a resolution

of 1 Da and a peak width of 0.75 Da.

Isoelectric point estimation by 2D-PAGE

Isoelectric focusing was performed on 7cm IPG ReadyStrips pH 3–10NL (Bio-Rad).

Concentrated protein (20 µg) was dissolved in a sample buffer containing 7 M urea, 2 M

thiourea, 4% (w/v) CHAPS, 0.005% (w/v) bromophenol blue, 0.5% (v/v) destreak reagent

(GE Healthcare, Uppsala, Sweden) and 0.5% ampholyte and incubated at room

temperature for 60 min. The first-dimension separation was performed on a Protean IEF

Cell System (Bio-Rad). The IPG strip was loaded using active in-gel rehydration.

Electrofocusing was performed as follows: 250 V for 15 min, then at a linear gradient from

250-4,000 V for 2 h, followed by 4,000 V until a total of 18,000 V/hr was reached. The

voltage decreased to a holding voltage of 500 V. After IEF, the strip was equilibrated with

a sample equilibration buffer (SEB) containing 6 M urea, 375 mM Tris-HCl (pH 8.8), 2%

(w/v) Sodium Dodecyl Sulphate (SDS), 30% (v/v) glycerol, and 0.0125% (w/v)

bromophenol blue. In the first incubation, SEB contained 1% (w/v) dithiothreitol, after 8

min this solution was replaced with SEB containing 25% (w/v) iodoacetamide and the strip

was incubated for a further 8 min. The IEF strip was placed onto 8% (v/v) polyacrylamide

gels and sealed in with agarose. SDS-PAGE was then carried out at 125 V for 50 min.

Proteins were visualized by staining with Imperial Protein Stain (Pierce Biotechnology)

and gel images were scanned on a GS800 scanner (BioRad). Precision Plus Protein

Standards (Bio-Rad) were used as molecular weight markers. A separate 2D-gel was

performed with 2-D SDS-PAGE Standards (15 µl) (Bio-Rad) to provide calibrated pI

points to allow estimation of the pI of PgChP.

RESULTS

Genetic characterisation of P. chrysogenum AS51D

Amplification of the 18S-28S rRNA intergenic spacer (ITS) sequence revealed a band of

585 bp, which was sequenced and compared against other sequences in BLAST. The strain

AS51D displayed 100% of identity and the maximum score with the 18S-28S rRNA

intergenic spacer of Penicillium chrysogenum strain FRR 807, accession number in

GenBank: AY373839. Thus, AS51D was confirmed as P. chrysogenum.

RESULTADOS

97

Analysis of PgChP protein by SDS-PAGE

SDS-PAGE of purified PgChP to estimate the molecular mass was revealed two discrete

bands (Figure 1): an intensely stained band showing a molecular mass of 37 kDa and a

fainter one of around 40 kDa.

Mass spectrometric analysis of PgChP digestions

Figure 2 shows typical survey spectrum peptide profiles obtained by mass spectometry for

the 37 kDa protein band digested with trypsin and chymotrypsin. The 40 kDa band was

also analysed in a similar manner. Overall, the peptide mass spectrum profiles obtained for

the upper and lower bands showed a high degree of similarity (Figure 3).

Figure 1. SDS-PAGE of PgChP protein, showing two bands of 40 kDa (1) and 37 kDa (2). M:

Pharmacia low molecular weight standard.

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MANUSCRITO 4

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Figure 2. Mass spectra showing typical peptide profiles obtained from trypsin (A) and

chymotrypsin (B) digestions of PgChP (37 kDa band).

RESULTADOS

99

De novo amino acid sequences

Tandem MS was also performed on peptides from PgChP trypsin and chymotrypsin

digestions to obtain amino acid sequences. All of the 40 kDa band peptide sequences

obtained were also present in the 37 kDa band. Together with the mass spectrum profiles,

the data indicated that the two bands observed on SDS-PAGE analysis were likely to

represent the same protein or two very similar proteins. The 37 kDa band was subjected to

extensive tandem MS analysis to provide increased amino acid sequence coverage of the

protein.

A total of twelve de novo peptide sequences were obtained from trypsin and chymotrypsin

digestions from the 37 kDa band and four from the 40 kDa band (Table 1).

Figure 3. Mass spectra showing the highly similar peptide profiles obtained from trypsin

digestion of the 37 kDa (A) and 40 kDa (B) bands from SDS-PAGE of PgChP.

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MANUSCRITO 4

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Table 1. PgChP-de novo peptide sequences obtained from trypsin and chymotrypsin digestions

from 37 kDa and 40 kDa. L and I are interchangeable. Italic letters indicate amino acids which

were tentative assignments.

*: Indicates sequences from the 40 kDa band.

-: indicates the presence of additional unassigned amino acids.

Sequence

Number de novo sequence

Peptide

mass Charge

Digestive

enzyme

1 NKPDGGPPGSYF 618.29 +2 Chymotrypsin

2a -FAAGSSLPVAALQSAAAK 959.81 +3 Trypsin

2b* YFAAGSSLPVAALQSAAAK 911.48 +2 Trypsin

2c* PVAALQSAAAK 1026.61 +1 Trypsin

3 -PDATYPLDGDNGA- 866.40 +2 Trypsin

4 -TIHSDWAK 528.79 +2 Trypsin

5 -WLADMDVDCDGLD- 1139.03 +2 Trypsin

6 GNPDGQHQTNFGAL- 728.33 +2 Trypsin

7 -AAYEVPFFVIPDR 988.21 +3 Trypsin

8a GALPGNNVGAVICDGK 771.40 +2 Trypsin

8b GALPGNNVGAVICDGK 720.89 +2 Chymotrypsin

9a -PQVLGEASWLMAR 1053.50 +2 Trypsin

9b* MFYGIYGDSDGDTPQVLGEASRFFAR 972.43 +3 Trypsin

10a FTGDDSVLPSSALNK 775.87 +2 Trypsin

10b* -TYILFTGDDSVLPSSALNK 1352.95 +3 Trypsin

11 NYVTNFTTLR 614.82 +2 Trypsin

12 RSMGDKLM 469.22 +2 Chymotrypsin

RESULTADOS

101


After enzymatic deglycosylation of PgChP with the PNGase F or the E-DEGLY

deglycosylation kit, only the band of 37 kDa was detected (Figure 4). However, chemical

deglycosylation with HCl and NaOH revealed no changes in spectra with untreated and

treated PgChP by ESI-MS.

Molecular mass determination of PgChP protein by electrospray ionization mass

spectrometry

ESI-MS was used to establish the accurate mass of intact PgChP. The m/z spectrum was

deconvoluted and converted to the molecular mass profile using Maximum Entropy

processing (Figure 5A). The mass profile shows a dominant peak of 30,771.9 Da with a

series of surrounding peaks differing from each other by approximately 162 Da (Figure

5B).

Figure 4. SDS-PAGE of purified PgChP untreated (lane 1), and treated with PNGase F (lane

2). The arrow indicates the 37 kDa band which remains after deglycosylation. M: Precision

Plus Protein Standard.

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MANUSCRITO 4

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Figure 5. Molecular mass profile from Maximum Entropy processing of PgChP. The most

abundant peak has a mass of 30771.9 Da (A). Detailed view revealing a series of peaks

separated by approximately 162 Da (B). Additional peaks surrounding the major peaks

represent sodium adducted forms (+/- 22 Da).

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RESULTADOS

103

Isoelectric point estimation

2D-PAGE analysis showed that PgChP migrates on a broad pH range gel in a discrete

region of the gel at approximately pH 5 (Figure 6).

DISCUSSION

In previous studies the strain AS51D was characterized as P. chrysogenum using methods

based on morphology and biochemical characteristics, including the production of

secondary metabolites by high performance liquid chromatography (Acosta et al., 2009a).

In the present study, the sequence of the 18S-28S rRNA intergenic spacer confirmed the

previous characterization. P. chrysogenum is regarded as a safe mould of starter cultures

(Núñez et al., 1996; Benito et al., 2003) together with other close species, such as

Penicillium nalgiovense (Geisen, 2000). To fulfill this role, AS51D could be used either as

a source for antifungal proteins or as protective culture.

Figure 6. Isoelectric point (pI) determination of PgChP by 2D SDS-PAGE. A: PgChP is

indicated with an arrow. B: 2D SDS-PAGE standards. Numbers within figure B indicate the pIs

of 4 standard proteins that migrate as a series of spots.

A)

B)

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MANUSCRITO 4

104

Two bands were found in the concentrated PgChP fraction by SDS-PAGE analysis with

estimated molecular weights of 37 and 40 kDa. The comparison between the amino acid

sequences obtained from both bands showed a high degree of similarity (Table 1). All

sequences obtained from the 40 kDa band were derived from three different regions of the

37 kDa protein: sequences 2a, 2b, and 2c in one region; 9a and 9b in a second; and 10a and

10b in a third region. Where they overlapped, peptide sequences from the 40 kDa band

were identical to those from the 37 kDa band, with the only exception being for peptide 9b

for which 3 of the 5 C-terminal residues could be only tentatively assigned from the

tandem MS spectrum. The higher number of sequences obtained from the 37 kDa band

result from the more in-depth analysis carried out with this protein, including digestion

with chymotrypsin, and in-gel and in-solution digestions.

After exposing the PgChP protein to N-Glycosidase F (PNGase F) only the protein of 37

kDa was observed (Figure 4). This deglycosylation enzyme cleaves between the innermost

GlcNAc and asparagine residues of high mannose hybrid and complex oligosaccharides

from N-linked glycoproteins (Maley et al., 1989; Plummer and Tarentino, 1991). Thus, the

protein of 40 kDa is glycosylated, and its peptidic core migrates at the same molecular

mass as the 37 kDa protein. Chemical deglycosylation with HCl and NaOH produced no

changes in mass spectra when treated and untreated PgChP were compared by ESI-MS.

Thus, neither sialic acid residues nor O-linked oligosaccharides are present (Leis et al.,

1997; Geert and Thomas-Oates, 1998; Krapf et al., 2006; Tarelli, 2007).

The analysis of the molecular mass of the protein by mass spectrometry revealed the

existence of six major peaks centring a dominant peak of around 30.8 kDa, differentiated

by a mass multiple of 162 Da, the mass of a galactose or mannose residue. It is therefore

possible that the purified fraction of PgChP has multiple forms of the protein each with a

different number of mannose or galactose residues. The glycosylation of the protein likely

explains the difference in the mass estimated by SDS-PAGE and ESI-MS. It is known that

glycoproteins can migrate atypically by SDS-PAGE because SDS does not bind

oligosaccharides (Nelson, 1971; Russ and Poláková, 1973; Bagger et al., 2007). Given that

the ESI-MS provides an exact molecular mass to within 0.01%, it can be deduced that the

protein has an atypical migration by SDS-PAGE.

Glycosylated proteins are not uncommon among antifungal enzymes, such as an antifungal

glycoprotein from Urginea indica (Deepak et al., 2003) or chitosanases from Mycobacter

AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton and Eveleigh, 1981).

RESULTADOS

105

Glycosylation represents the most common form of protein post-translational

modifications. Carbohydrates can be found in both eukaryotes and prokaryotes and they

participate in many biological processes including inter- and extra-cellular signalling,

protein regulations and interactions or molecular recognition. Glycan chains are added to

proteins as a set of variations on a core structure, and can vastly increase the complexity of

protein molecules. Glycans can determine the localization, activity and function of proteins

and can influence physicochemical characteristics of proteins such as folding, solubility,

resistance to degradation by proteases or increase in thermal stability (Hildegard and

Geyer, 2006; Morelle et al., 2006).

In N-glycosylation, glycans are attached to the protein via N-acetylglucosamine (GlcNAc)

to the amide group of asparagine within an Asn-X-Ser/Thr (sometimes also Cys) motif,

where X is any amino acid apart from proline (Morelle et al., 2006). Although the

complete amino acid sequence of PgChP is unknown, at least one consensus site for N-

glycosylation is present in the protein (Table 1, peptide 11 -NFT-). This potential site for

glycosylation in PgChP supports the interpretation that the 37 kDa band represents the

deglycosylated core of the 40 kDa band.

The de novo amino acid sequences obtained from PgChP (Table 1) showed the highest

similarity with sequences of seven described or predicted fungal chitosanases from A.

fumigatus (accession number: XP_754126), N. fischeri (XP_001262949), A. niger

(XP_001390407), A. oryzae-1 (BAD08218), A. oryzae-2 (XP_001824257), A. terreus

(XP_001209034), and A. clavatus (XP_001274664). The twelve peptide sequences from

PgChP were placed onto a multiple sequence alignment of these protein sequences (Figure

7).

Three highly conserved amino acid sequences have been described in all of the fungal

chitosanases. These three regions are located in the vicinity of the centre of the open

reading frames, and are considered to be important for catalytic function (Shimosaka et al.,

2005). In each of these three regions there is one conserved carboxylic amino acid (Glu or

Asp) considered to be essential or important for the catalytic function as a proton donor in

a large number of glycosyl hydrolases (Monzingo et al., 1996; Robertus et al., 1998). At

least one of these conserved regions corresponds with the de novo peptide sequence 9 of

PgChP, which has the conserved glutamic acid (underlined): PQVLGEASWLMAR.

MANUSCRITO 4

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Figure 7. Alignment of PgChP amino acid sequences with the most similar proteins in the public

sequence databases. Shaded-background letters show similarities of the proteins with the obtained

sequences of PgChP. Accession numbers in Genbank are XP_754126 (A. fumigatus),

XP_001262949 (N. fischeri), XP_001390407 (A. niger), BAD08218 (A. oryzae-1), XP_001824257

(A. oryzae-2), XP_001209034 (A. terreus), and XP_001274664 (A. clavatus). Asterisks indicate

amino acid residues completely conserved in all sequences. Numbered segments correspond to de

novo obtained peptide sequences for PgChP (see Table 1).

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* *** * * ***** * * PgChP NKPDGGPPGSYFAAGSSLPVAALQSAAAK A.fumigatus MPSTTIIRQLAISLALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK N.fischeri MPSKTIIRQLAISVALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK A.niger MAFKTTAG--LAFLALAG-SVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAAK A.oryzae-1 MPIKSFASRLALSLAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK A.oryzae-2 MPIKSFASRLALSLAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK A.terreus MVFKKAAIGLTLPFALFSSVALGQTVDGSDYDSPNGGPPGSYFAAASTMPVAALQAAAAK A.clavatus MIFKSSLSHAAASLLLLT-PALAQKVQGPEYNKPSAGPPASFFAAAPTMPVAALKSAVAR * ** *** ** ********** PgChP PDATYPLDGDNGA TIHSDWAK WLADMDVDCDGLD A.fumigatus ATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------- N.fischeri ASKVPSLATYPVSQDSGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------- A.niger ASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYKC------- A.oryzae-1 ASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------- A.oryzae-2 ASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------- A.terreus ATKVPSYATYPVSQDDNAKKSTIHSDWASFSQGAAISWVADMDVDCDGIDSGCEHEIKTS A.clavatus ASVVPKNAAYPVNQD-GGPTATIHADWASLPTAAAYVYTADMDVDCDGLDHNC------- ** ** ** *** ***** *** * ** **** ** PgChP GNPDGQHQTNFGALAAYEVPFFVIPDR AGALPGNNVGAVIC----DGK A.fumigatus QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANSGALPGNNIAAVIC----NGKMFYGIL N.fischeri QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANTGALPGNNIAAVIC----NGKMFYGIL A.niger KGNGDGLPETNWGALSAYEVPWIVIPDQFLTANEDLLPGNNVAAVIC----NGKMYYGIL A.oryzae-1 KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVIC----NGKMFYGIF A.oryzae-2 KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVICATSSNGKMFYGIF A.terreus QGNPDGQDATNWGALAAYEVPFIVIPQKYLDHNGNALKGNNIAAVIC----NGKMFYGIL A.clavatus KGNPDGQPDTNFGALAAYEVPFVVIPDRFATTYASALPGNNIVAVIC----DGKMFYGIF * * ********* * ** ** PgChP PQVLGEASWLMAR LLFTGDDSVLPSSALNKNY A.fumigatus GDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALTKNY N.fischeri GDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKDAVLPSSALTKNY A.niger GDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALNKNY A.oryzae-1 GDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKNY A.oryzae-2 GDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKNY A.terreus GDANGDEPQVTGEASWLMARTCFPNEGLNGNKGHTAADVTYILFTGDESVLPSSALNENY A.clavatus GDTDGDHPQVIGEASWLMARTCFPNDNLNGDSGHVPADVTYIFFTGKDSVLPSSAVNKNY * * ****** * PgChP VTNFTTLRSMGDKLM A.fumigatus ITNFTTLRSMGDKLVNALVSNLGLSGTPPTK---------TTTRVTTTTTKPT-SAASCS N.fischeri ITNFTTLRSMGDKLVNALASKLGLSGTPPTK---------TTTLVTTTTTKPT-STASCS A.niger ITNFSTLRSMGDKLVGALASNLGLTSSA------------------------SGSTATCS A.oryzae-1 ITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSCS A.oryzae-2 ITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSCS A.terreus ITNFSTLRSMGDKLVNALASNLGLSGGSGGT---------PTT--TTTATQPT-STGTCS A.clavatus ITDFTKLRSMGDSLVNAFASQLGISSGGGNGGGDGGHTTLHTTTATRTATAPT-STATCD PgChP A.fumigatus WAGHCL-----GASCSSDDDCADALVCTAGKCSVDGAA-TCSWEGHCEGASCSSDDDCSD N.fischeri WAGHCL-----GASCSSNDDCADALVCAAGKCSVDGAA-TCSWEGHCEG----------- A.niger WQGHCE-----GAICSTEDDCSDDLVCDSGKCSSPDED---------------------- A.oryzae-1 WPGHCA-----GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCSD A.oryzae-2 WPGHCAGFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCSD A.terreus WAGHCE-----GASCSTNDDCSDQLACKNGVCSTDGEV-VCSWEGHCEGATCSSNDDCSD A.clavatus WEGHCL-----GTACSNNGECSDPFGCINGFCNYDPTL-TCQWAGHCVGASCSSNDQCSD PgChP A.fumigatus DLYCKSGSCTA------------------------------------P--- N.fischeri --------------------------VYS---------------------- A.niger ----------------DGDDEDDEDEEENDEDDEDDDDEDDEDDE------ A.oryzae-1 ELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- A.oryzae-2 ELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- A.terreus ELYCAGGACTSA--------------------------------------- A.clavatus PFACIDGACAVDT-SLDCSRKGHCAGTTCLSDSDCSRPLSCILGVCANQSG

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RESULTADOS

107

A protein sequence homology analysis was performed calculating the percentages of

amino acid identity between the sequences obtained from PgChP protein shown in table 1

and the aligned sequences from the seven fungal chitosanases (Table 2).

Table 2. Similarity between the amino acid sequences obtained from PgChP and the aligned amino

acid sequences of the similar fungal chitosanases described in Figure 7. Percentages higher than

85% are background shaded.

All peptide sequences from PgChP showed high similarity (from 38 to 92%) with the

fungal chitosanases (Table 2). In addition to sequence 9, already discussed as a conserved

region in fungal chitosanases, sequence 5 had a similarity higher than 85% in all the

compared proteins.

The high similarity with fungal chitosanases and the conserved region identified in

sequence 9, confirm that PgChP belong to this kind of proteins. Chitosanases (E.C.

3.2.1.132) are the glycoside hydrolases that cleave the glycosidic bonds of chitosan. These

enzymes have been reported from viruses, bacteria and fungi (Wang et al., 2008).

Chitosanases are classified into five glycoside hydrolase families, GH-5, GH-8, GH-46,

GH-75, and GH-80, according to amino acid sequence homology. Fungal chitosanases

Similarity (%)

PgChP

SequencesA. fumigatus N. fischeri A. niger A. oryzae-1 A. oryzae-2 A. terreus A. clavatus

1 75 75 67 75 75 75 67

2a 78 78 83 83 83 78 61

2b* 74 74 84 79 79 79 53

3 61 54 38 46 46 46 38

4 75 75 75 75 75 75 75

5 85 85 92 85 85 92 85

6 86 86 64 79 79 79 86

7 77 77 69 69 69 77 92

8a,b 76 76 71 65 65 65 82

9a 92 92 85 92 92 92 92

9b* 73 73 69 73 73 69 73

10a 72 78 67 83 83 83 78

10b* 76 81 43 86 86 86 81

11 90 90 80 80 80 80 70

12 87 87 87 75 75 87 75

MANUSCRITO 4

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belong to the GH-75 family (Cheng et al., 2005), which is composed mainly by

chitosanases from Aspergillus (Cheng and Li, 2000; Zhang et al., 2000) and Fusarium

(Shimosaka et al., 1996; Cantarel et al., 2008). Most chitosanases are characterized by a

low molecular mass in the range of 10-50 kDa (Fenton and Eveleigh, 1981; Somashekar

and Joseph, 1992; Alfonso et al., 1992).

The isoelectric point (pI) estimated from 2D-PAGE and isoelectrofocusing was close to pH

5, which is within the range of isoelectric points, from pH 4 to 5, described for fungal

chitosanases (Fenton and Eveleigh, 1981; Alfonso et al., 1992; Shimosaka et al., 1993).

The range of predicted isoelectric points (pH 3.83-4.97) and molecular masses (33-40 kDa)

of the selected fungal chitosanases are quite similar to the experimental values obtained for

PgChP (Table 3).

Table 3. Isoelectric points (pI) and molecular masses of PgChP and selected fungal chitosanases.

* Predicted pI values and theoretical molecular masses were calculated using the ProtParam tool in

the ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org).



CAP80409) that shows high homology with the amino acid sequences obtained from

PgChP in this work. However, the production of the protein by this mould has not been

reported.

Currently, only two chitosanases have been reported from the genus Penicillium, one from

P. spinulosum and another from P. islandicum (Fenton et al., 1981; Ak et al., 1998), but

none from P. chrysogenum. Given that P. chrysogenum is used to obtain generally

Isoelectric Point Molecular Mass (kDa)

Protein/producer mould 2D-PAGE/IEF Predicted* ESI-MS Theoretical*

PgChP/ P. chrysogenum 5 - 30-31 -

XP_001274664/ A. fumigatus - 4.64 - 35.1

XP_001262949/ N. fischeri - 4.97 - 33.1

XP_001390407/ A. niger - 3.83 - 33.4

BAD08218/ A. oryzae-1 - 4.69 - 38.9

XP_001824257/ A. oryzae-2 - 4.71 - 39.8

XP_001209034/ A. terreus - 4.25 - 36.0

XP_001274664/ A. clavatus - 4.64 - 40.1

RESULTADOS

109

recognized as safe (GRAS) enzymes, PgChP could be used as a food preservative against

toxigenic moulds. However, before using PgChP for food protection, an in-depth

toxicological evaluation of the antifungal protein is a prerequisite for its application. Thus,

a full characterization should be done for this novel chitosanase.

ACKNOWLEDGEMENTS


06546-ALI) and FEDER. Andrea Rodríguez was the recipient of a FPI grant from the

Spanish Ministry of Education and Science. Thanks to Alex Rathbone, University of

Nottingham, for tuition in 2D gels.

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Manuscrito 5: Genetic characterization of

the gene enconding the novel chitosanase PgChP


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Genetic characterization of the gene encoding the novel chitosanase

PgChP from Penicillium chrysogenum

Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa, Miguel A.

Asensio*


Universidad, s/n. 10071- Cáceres, Spain. a Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de

Cáceres s/n, 06071- Badajoz, Spain



ABSTRACT

The protein PgChP is a new chitosanase with antifungal activity produced by Penicillium

chrysogenum AS51D. The gene encoding the chitosanase PgChP has been sequenced. A

DNA fragment of the gene encoding pgchp was obtained by PCR using degenerate primers

based on two known amino acid sequences of the purified PgChP. cDNA was prepared for

RACE-PCR from total RNA of P. chrysogenum by RT-PCR. Full-length cDNA and

complete genomic DNA of pgchp were obtained using primers designed from 5’ and 3’ –

ends. Comparing the cDNA and genomic DNA, four introns of 60, 53, 59, and 59 bp were

observed. Full-length cDNA has 915 bp, corresponding to an open reading frame encoding

a protein of 304 amino acid residues. The first 21 amino acids matched a signal peptide.

The predicted mass of the translated nucleotidic sequence was 32 kDa and the predicted

isoelectric point was 4.8. The translated sequence of pgchp revealed that the protein had

two carboxylic amino acids (Glu and Asp) described as essential for the catalytic function

of fungal chitosanases. Two N-glycosylation consensus sequences were observed in the

translated pgchp gene. The putative amino acid sequence showed high homology with

fungal chitosanases.

KEY WORDS: antifungal activity, glycosylation, N-linked oligosaccharides, dry-

fermented foods.

INTRODUCTION

P. chrysogenum AS51D was isolated from meat products in previous studies (Acosta et al.,

2009a). This mould produces the protein PgChP, selected for its ability of inhibiting the

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growth of some toxigenic moulds. In previous works, different de novo amino acid

sequences obtained from PgChP showed significant similarities with the respective

sequences of seven fungal chitosanases, belonging to the GH-75 family of fungal

chitosanases (Rodríguez-Martín et al., 2009). Chitosanases (E.C. 3.2.1.132) are produced

by a large number of microorganisms including bacteria and fungi. Numerous bacterial

chitosanases are able to degrade exogenously added chitosan (Shimosaka et al., 2000;

Yoon et al., 2002; Lee et al., 2006; Zhang et al., 2007). On the contrary, fungal

chitosanases have been reported from only a limited number of strains and their

physiological roles are unclear (Fenton and Eveleigh, 1981; Zhang et al., 2000). The

synthesis of some chitosanases can be induced by adding different substrates such as

glucosamine (Zhang et al., 2001) and chitosan (Zhu et al., 2007).

Analysis of the molecular weight of PgChP by ESI-MS revealed six isoforms differing

from each other by approximately 162 Da, the mass of one residue of mannose. These

evidences indicated that the chitosanase PgChP was a glycoprotein with different degrees

of glycosylation. Various deglycosylation treatments revealed the presence of N-linked

oligosaccharides in PgChP (Rodríguez-Martín et al., 2009).

The aim of this paper was to study the genetic sequence encoding the chitosanase PgChP.

This sequence will allow studying the existence of introns, the complete putative amino

acid sequence, and glycosylation consensus sequences.


Microbial strain

P. chrysogenum AS51D was isolated from dry-cured ham and belongs to the fungal

collection of Food Hygiene, University of Extremadura.

DNA isolation

P. chrysogenum AS51D was grown in malt extract broth made with 2% (w/v) glucose, 2%

(w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking, for

5 days. The mycelium was obtained by filtering the culture through paper Whatman nº2.

Two grams of mycelium were broken with mortar and pestle after freezing by adding

liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml

0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl

RESULTADOS

117

(TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Next, 200 µl proteinase K (20

µg/µl) were added, and the mix was incubated at 60ºC for 35 min, cooled on ice and

extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was

centrifuged at 3000×g for 2 min at 4ºC. The upper phase containing DNA was transferred

to a fresh tube and precipitated by adding 3 M sodium acetate pH 5.2 to a final

concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the

pellet was cleaned with 70% (v/v) ethanol, centrifuged again, resuspended in sterile water,

and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted

with phenol-chloroform-isoamyl alcohol, and precipitated again as indicated above,

resuspended in water, and stored at -70ºC. Quantity and quality of purified DNA was

determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg,

Germany).

Genomic DNA amplification

Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50

µl reaction mixtures containing 50 pmol each of forward and reverse degenerated primers,

0.5 mM each of deoxyribonucleotide triphosphates, (dNTPs, Roche, Madrid, Spain), 0.1

vol of 10× PCR buffer (22.5 mM MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium

sulfate), 1.8 mM MgCl2 and 2.5 units of pfu turbo polymerase (Stratagene, La Jolla, USA).

The degenerated primers pgchp-DPF1 and pgchp-DPR2A (Table 1) were designed from

amino acid sequences previously obtained by mass spectrometry (Rodríguez-Martín et al.,

2009). Additionally, for pgchp-DPR2A the contiguous residues conserved in fungal

chitosanases (Shimosaka et al., 2005) were also included.

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DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific,

Cramlington, UK). Three reactions were done, one with both forward and reverse primers,

and two with either forward or reverse primers. The PCR program consisted of initial

denaturation (94ºC for 5 min), 35 cycles of denaturation (94ºC for 1min), annealing (56-

60ºC range for 30 s, gradient 12), and extension (72ºC for 30 s), followed by a final

extension at 72ºC for 4 min. PCR products were electrophoresed on 2% (w/v) agarose gels

in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA), pH 8 ussing a Sub-Cell GT

apparatus (Bio-rad Laboratories, Madrid, Spain). Two different markers were used for

agarose gels: 1kb DNA ladder (0.25-12 kb bands) (Promega, Hampshire, United Kingdom)

and DNA size standard (0.05-2kb) (Bio-Rad). Then, products detected only in the reactions

with both reverse and forward primers were gel-purified and cloned into the pCR2.1-

TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10 Chemically Competent E.

coli cells (Invitrogen) were transformed with that vector. To confirm the insert in the

vector, digestions with restriction enzyme EcoRI (Roche) were carried out. Finally, vectors

were sequenced in the Institute of Biomedicine (CSIC, Valencia, Spain).

RNA isolation

P. chrysogenum AS51D was grown in malt extract broth, pH 4.5 at 25ºC under continuous

shaking for 4 days. Chitosanase production was induced in a medium with glucosamine

(Zhang et al., 2001) made with 0.3% (w/v) NaNO3, 0.05% (w/v) KCl, 0.05% (w/v)

MgSO4•7H2O, 0.01% (w/v) FeSO4•7H2O, and 2% (w/v) D(+)-glucosamine hydrochloride

(Sigma, Dorset, United Kingdom), dissolved in 25 mM phosphate buffer pH 5.8. Mould

Table 1. Degenerated primers used to amplify the pgchp gene.

Primer names Amino acid sequences and designed primers*

pgchp-DPF1

N K P D G G P

5’-AAY AAR CCN GAY GGN GGN CC-3’

pgchp-DPR2A

M A R T C F P

3’-AC CGN GCN TGN ACR AAR GG-5’ * R for purine, Y for pirimidine, N for A, C, T, or G.

Conserved amino acids used for designing the primer pgchp-DPR2A are underlined.

RESULTADOS

119

was cultured in glucosamine medium at 25ºC under continuous shaking for 17 h.

Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until

RNA extraction. Frozen mycelium was broken with mortar and pestle after adding liquid

nitrogen. RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen, Crawley,

UK) following the manufacturer’s instructions. Total extracted RNA was resuspended in

40 µl of RNase-free deionized water.

Rapid amplification of cDNA ends by Polymerase Chain Reaction (RACE-PCR)

Amplification of 5’ and 3’ ends of the pgchp gene was carried out using the SMART

RACE cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye,

France).

cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two

separate populations of cDNA were synthesized according to the amplification kit

instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the

SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR

reactions were performed using the Universal Primer A Mix (UPM), in conjunction with

distinct gene-specific primers.

Gene-specific primers design. Two gene-specific primers (GSP) were designed for

amplifying the 5’ and 3’- ends (5’-RACE GSP: CCATTGCAGATAACGGCACCAACG

and 3’-RACE GSP: GTTCGACAAGGGTGCGGCATATG). Polymerase chain reactions

were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as template in 50 µl reaction

mixtures containing 1 mM of dNTPs (Roche), 0.5 µM of the 5’- or 3’-RACE GSP primers,

5 µl of Universal Primer A Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH

8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, and 2.5 units pfu turbo polymerase

(Stratagene). A program for touchdown PCR was carried out for 5’-end amplification: 5

cycles of 2 steps (94ºC for 30 s and 72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s,

70ºC for 30 s and 72ºC for 3 min) and 27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s

and 72ºC for 3 min). The PCR program to obtain the 3’-end consisted of 30 cycles of

denaturation (94ºC for 30 s), annealing (68ºC for 30 s), and extension (72ºC for 3 min).

Finally, 5’- and 3’-RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO

vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli

(Invitrogen). Then plasmids were purified and sequenced. Digestions with restriction

enzyme EcoRI (Roche) were performed to confirm the insert in the vector.

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120

Full-length cDNA amplification. The full cDNA fragment of the gene pgchp was obtained

by PCR with specific primers Fwd-pgchp (ATGATGACCTACAGCCGCTTAATCCC)

and Rev-pgchp (TCAATCACTGGGGCATTTTCCAC) designed from 5’ and 3’ ends.

PCR reaction mixtures at a final volume of 50 µl contained 5’-RACE-Ready cDNA (50

ng), 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of

10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM

MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program consisted of 30

cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3

min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These

plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells

(Invitrogen). Then vectors were purified and sequenced. Digestions with restriction

enzyme EcoRI (Roche) were performed to confirm the wanted insert in the vector.

Amplification of the whole genomic gene

The genomic gene sequence was amplified by PCR using Fwd-pgchp and Rev-pgchp

primers. PCR reactions contained 100 ng of genomic DNA, 0.5 µM each of forward and

reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl,

pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase

(Stratagene), and deionized water until a final volume of 50µl. The PCR program consisted

of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC

for 3 min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These

plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells

(Invitrogen). Then vectors were purified and sequenced. Alignment between gene

sequences obtained from genomic DNA and cDNA was performed. Introns sequences

were confirmed by GenScan software.

RESULTS

Partial amplification of pgchp gene using degenerated primers

The pgchp gene partial amplification from genomic DNA was done using two degenerated

primers: pgchp-DPF1 and pgchp-DPR2A (Table 1). After different reactions, the optimal

annealing temperature was 57.7ºC. PCR products obtained with these primers were run in

RESULTADOS

121

agarose gel. Just one band with high intensity was amplified in reactions with both forward

and reverse primers (Figure 1). This band was cloned and sequenced.

After sequencing, the selected fragment was found to consist of 657 bp. The obtained

sequence was translated and compared in Basic Local Alignment Search Tool (BLAST)

with known sequences. A high degree of similarity with several fungal chitosanases was

found, as it is discussed later. The common sequences included three introns described for

genes encoding fungal chitosanases.

Sequence of the pgchp gene by RACE PCR

After induction with glucosamine, mRNA was extracted and used to obtain both 5’- and

3’-RACE-Ready cDNA populations by RT-PCR. Two gene specific primers were

designed from the obtained genomic DNA sequence without introns to get 5’- and 3’-ends:

5’-RACE GSP (5’-CCATTGCAGATAACGGCACCAACG-3’) and 3’-RACE GSP (5’-

GTTCGACAAGGGTGCGGCATATG-3’), respectively. After 5’ and 3’-RACE reactions,

two bands with the expected size (Figure 2) were gel-purified from 5’- and 3’-RACE

Figure 1. PCR products using forward DPF1 (F), both forward DPF1 and reverse DPR2A (F+R),

and reverse DPR2A (R) primers. M: DNA standards. The arrow indicates the selected band.

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MANUSCRITO 5

122

PCRs, cloned, and sequenced. The cDNA ends of the gene 5’ and 3’ were 559 bp and 796

bp, respectively.

Next, two new primers were designed to obtain the full encoding cDNA (Fwd-pgchp: 5’-

ATGATGACCTACAGCCGCTTAATCCC-3’ and Rev-pgchp: 5’-

TCAATCACTGGGGCATTTTCCAC-3’). The full-length cDNA contained a 915 bp open

reading frame encoding a protein of 304 amino acid residues (Figure 3) that included the

known PgChP sequences.

Genomic DNA of pgchp gene, amplified using the same primers for cDNA, was 1146 bp

length. Comparison of cDNA and genomic sequences revealed that pgchp gene had four

introns with a length of 60, 53, 59, and 59 bp (Figure 3). These introns were confirmed by

GenScan program.

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RESULTADOS

123

The open reading frame was translated to protein by ExPASy proteomics server at the

Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase

cleavage site predicted by the SignalP program (Nielsen et al., 1997) suggested that the

pre-sequence consisted of the first 21 amino acids (MMTYSRLIPFALFALFGTGLA).

5’-atgatgacctacagccgcttaatccccttcgctctctttgccttgtttggcacagggctc M M T Y S R L I P F A L F A L F G T G L gcgcaaacagtcgatggctccaaattcaacaagccagacggcggtccaccaggaagctac A Q T V D G S K F N K P D G G P P G S Y ttcgctgctggctcgtctattcccgtcgctgctttgcagagcgctgctgcaaaggctcgc F A A G S S I P V A A L Q S A A A K A R actcccgtgccagatgcaacctatcctattaatggcgataacggtgctaagaaagttacc T P V P D A T Y P I N G D N G A K K V T atccacagcgactgggctaagttcgacaaggtagatactctcactggtcggttaattgca I H S D W A K F D K << Intron 1 aggtccgcatgcttattaaatggatcatagggtgcggcatatgtttggattgcggatatg >> G A A Y V W I A D M gacgtcgactgcgacggcattgactacaagtgcaaggtacggtcactattgttgtctgtc D V D C D G I D Y K C K << Intron 2 tgagatgttattgctgacccgatgttcagggaaacccggatggtcagcaccaaaccaact >> G N P D G Q H Q T N tcggagctttggctgcgtatgaagtgccgttctttgtgattcccgacaggtttggaacca F G A L A A Y E V P F F V I P D R F G T agtacgcgaagcagcttcctggaaacaacgttggtgccgttatctggtatggtttcctac K Y A K Q L P G N N V G A V I C<< ctttgtaagatatctcagcggcgtcttgttctaaccgaaagctagcaatggaaagatgtt Intron 3 >> N G K M F ctacggaatttacggagactccgatggcgatacccctcaggtcatcggcgaggcctcatg Y G I Y G D S D G D T P Q V I G E A S W gttaatggcccggacctgcttccctaatgatgacttgaatggcaatagtggccatggtga L M A R T C F P N D D L N G N S G H G D tgttgatgtgacctgtaagtttgcagccttggcacatctagatccgtattctgtcaacta V D V T << Intron 4 ctaatctaaccagatatcctcttcactggcgacgactcggtgctccccagcagcgctctc >>Y I L F T G D D S V L P S S A L aacaagaattatgtcaccaacttcaccactctgcgctctatgggagacaaactcatgact N K N Y V T N F T T L R S M G D K L M T gctcttgcgaagaacctcaagttggtagatggaggtgatggaggttctccaacaactacg A L A K N L K L V D G G D G G S P T T T gctgggtccaaccctactagcgggtcttgtgagtgggagggacactgtgctggtgcgtct A G S N P T S G S C E W E G H C A G A S tgcaaagatgaaaatgattgctccgatcaactggtctgcaaaagtggaaaatgccccagt C K D E N D C S D Q L V C K S G K C P S gattga-3’ D -

Figure 3. Full-length DNA sequence of pgchp gene. The open reading frame has been

translated to amino acids. Typical sequences of fungal introns are underlined.

MANUSCRITO 5

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DISCUSSION

RACE-PCR is a method widely used in the characterization of genes. The successful of

this technique is highly depended on the amount of mRNA from the gene enconding the

studied protein. Thus, the production of PgChP was induced by glucosamine, as it has been

reported for other chitosanases (Zhang et al., 2001). After the mRNA extraction, both

populations 5’- and 3’-RACE-Ready cDNAs were satisfactorily obtained by RT-PCR.

Using gene specific primers designed from the sequence amplified with degenerated

primers, the 5’- and 3’-ends were obtained by RACE-PCR.

When the complete sequence was obtained, a difference between the pgchp gene from

genomic DNA (1146 bp) and from cDNA (915 bp) could be observed (Figure 3). Thus, the

existence of four introns of 60, 53, 59, and 59 bp in the gene, which are absent in the

cDNA, was confirmed by GenScan software. The small size of these introns compared to

mammalian introns is a typical feature from fungal genes (Gurr et al., 1987). The 5’- and

3’-ends of the four introns are very similar (Figure 3) and coincide with the consensus

splice sequences for fungal introns 5’-splice donor site (GT), the 3’-splice acceptor site

(AG). In addition, three of them have the internal CTRAC sequence (Figure 3), typical of

fungal introns (Wiesner et al., 1988).

The translated sequence of the gene pgchp encodes a chitosanase of 304 amino acids of the

PgChP protein. Comparing the putative amino acid sequence of pgchp gene with seven

fungal chitosanases, the homology was about 60 %. Several common sequences were

found among all these proteins (Figure 4). Three conserved regions have been described in

fungal chitosanases as important for catalytic function (Shimosaka et al., 2005). In two of

these regions there are one aspartic and one glutamic acid residues, essentials for catalytic

function of the chitosanase from F. solani. The predicted amino acid sequence of PgChP

also shows these two residues (Figure 4). Carboxylic amino acids (Glu or Asp) have been

identified as necessary residues for the catalytic function as proton donors in a large

number of glycosyl hydrolases (Monzingo et al., 1996; Robertus et al., 1998). PgChP and

the other seven fungal chitosanases show eleven common residues of aspartic acid as well

as two of glutamic acid (Figure 4). Moreover, 10 cysteines of PgChP are found in all these

fungal chitosanases (Figure 4). Cysteine residues are known to play an important role on

the stabilization of the tertiary structure by the formation of disulfide bridges in fungal

proteins (Nakaya et al., 1990; Lacadena et al., 1995; Marx et al., 1995; Lee et al., 1999).

RESULTADOS

125

PgChP --MMTYSRLIPFALFALFG-TGLAQTVDGSKFNKPDGGPPGSYFAAGSSIPVAALQSAAA 57 A.fumigatus MPSTTIIRQLAIS-LALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA 58 N.fischeri MPSKTIIRQLAIS-VALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA 58 A.niger MAFKTTAG--LAF-LALAG-SVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAA 56 A.oryzae-1 MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA 58 A.oryzae-2 MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA 58 A.terreus MVFKKAAIGLTLP-FALFSSVALGQTVDGSDYDSPNGGPPGSYFAAASTMPVAALQAAAA 59 A.clavatus MIFKSSLSHAAAS-LLLLT-PALAQKVQGPEYNKPSAGPPASFFAAAPTMPVAALKSAVA 58 PgChP KARTPVPDATYPINGDNGAKKVTIHSDWAKFDKGAAYVWIADMDVDCDGIDYKC------ 111 A.fumigatus KATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------ 112 N.fischeri KASKVPSLATYPVSQDSGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------ 112 A.niger KASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYKC------ 110 A.oryzae-1 KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------ 110 A.oryzae-2 KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------ 110 A.terreus KATKVPSYATYPVSQDDNAKKSTIHSDWASFSQGAAISWVADMDVDCDGIDSGCEHEIKT 119 A.clavatus RASVVPKNAAYPVNQD-GGPTATIHADWASLPTAAAYVYTADMDVDCDGLDHNC------ 111 PgChP -KGNPDGQHQTNFGALAAYEVPFFVIPDRFGTKYAKQLPGNNVGAVIC----NGKMFYGI 166 A.fumigatus -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANSGALPGNNIAAVIC----NGKMFYGI 167 N.fischeri -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANTGALPGNNIAAVIC----NGKMFYGI 167 A.niger -KGNGDGLPETNWGALSAYEVPWIVIPDQFLTANEDLLPGNNVAAVIC----NGKMYYGI 165 A.oryzae-1 -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVIC----NGKMFYGI 165 A.oryzae-2 -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVICATSSNGKMFYGI 169 A.terreus SQGNPDGQDATNWGALAAYEVPFIVIPQKYLDHNGNALKGNNIAAVIC----NGKMFYGI 175 A.clavatus -KGNPDGQPDTNFGALAAYEVPFVVIPDRFATTYASALPGNNIVAVIC----DGKMFYGI 166 PgChP YGDSDGDTPQVIGEASWLMARTCFPNDDLNGNSGHGDVDVTYILFTGDDSVLPSSALNKN 226 A.fumigatus LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALTKN 227 N.fischeri LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKDAVLPSSALTKN 227 A.niger LGDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALNKN 218 A.oryzae-1 FGDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN 225 A.oryzae-2 FGDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN 229 A.terreus LGDANGDEPQVTGEASWLMARTCFPNEGLNGNKGHTAADVTYILFTGDESVLPSSALNEN 235 A.clavatus FGDTDGDHPQVIGEASWLMARTCFPNDNLNGDSGHVPADVTYIFFTGKDSVLPSSAVNKN 226 * * PgChP YVTNFTTLRSMGDKLMTALAKNLKLVDGGDGG---------SPT--TTAGSNPT--SGSC 273 A.fumigatus YITNFTTLRSMGDKLVNALVSNLGLSGTPPTK---------TTTRVTTTTTKPT-SAASC 277 N.fischeri YITNFTTLRSMGDKLVNALASKLGLSGTPPTK---------TTTLVTTTTTKPT-STASC 277 A.niger YITNFSTLRSMGDKLVGALASNLGLTSSA------------------------SGSTATC 254 A.oryzae-1 YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSC 276 A.oryzae-2 YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSC 280 A.terreus YITNFSTLRSMGDKLVNALASNLGLSGGSGGT---------PTT--TTTATQPT-STGTC 283 A.clavatus YITDFTKLRSMGDSLVNAFASQLGISSGGGNGGGDGGHTTLHTTTATRTATAPT-STATC 285 PgChP EWEGHCA-----GASCKDENDCSDQLVCKSGKCPSD------------------------ 304 A.fumigatus SWAGHCL-----GASCSSDDDCADALVCTAGKCSVDGAA-TCSWEGHCEGASCSSDDDCS 331 N.fischeri SWAGHCL-----GASCSSNDDCADALVCAAGKCSVDGAA-TCSWEGHCEGVYS------- 324 A.niger SWQGHCE-----GAICSTEDDCSDDLVCDSGKCSSPDED--------------------- 288 A.oryzae-1 SWPGHCA-----GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCS 331 A.oryzae-2 SWPGHCAGFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCS 340 A.terreus SWAGHCE-----GASCSTNDDCSDQLACKNGVCSTDGEV-VCSWEGHCEGATCSSNDDCS 337 A.clavatus DWEGHCL-----GTACSNNGECSDPFGCINGFCNYDPTL-TCQWAGHCVGASCSSNDQCS 339 PgChP ---------------------------------------------------- A.fumigatus DDLYCKSGSCTA------------------------------------P--- 344 N.fischeri ---------------------------------------------------- A.niger -----------------DGDDEDDEDEEENDEDDEDDDDEDDEDDE------ 317 A.oryzae-1 DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- 380 A.oryzae-2 DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- 389 A.terreus DELYCAGGACTSA--------------------------------------- 350 A.clavatus DPFACIDGACAVDT-SLDCSRKGHCAGTTCLSDSDCSRPLSCILGVCANQSG 390

Figure 4. Alignment of PgChP with other fungal chitosanases. Common amino acids are denoted

by background shaded letters. Asterisks indicate essential amino acid residues for catalytic activity

of fungal chitosanases. Accession numbers in Genbank are XM_754126 (A. fumigatus),

XM_001262949 (N. fischeri), XM_001390407 (A. niger), BAD08218 (A. oryzae-1),

XP_001824257 (A. oryzae-2), XM_001209034 (A. terreus), XM_001274664 (A. clavatus).

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Six glycosylated isoforms of PgChP with N-linked oligosaccharides have been reported

(Rodríguez-Martín et al., 2009). In N-glycosylations, glycans are attached to the protein

via N-acetylglucosamine (GlcNAc) to the amide group of asparagine within an Asn-X-

Ser/Thr/Cys motif, where X is any amino acid apart from proline (Morelle et al., 2006).

Two N-glycosylation consensus sequences were found in the translated pgchp gene: NFT

(residues 230-232) and NDC (residues 288-290).

Glycosylation is the most common post-translational modification in proteins.

Carbohydrates participate in important biological processes, including inter- and

extracellular signalling, protein regulations and interactions, and molecular recognition.

Glycans can determine the localization, activity and function of proteins, as well as

influence on physicochemical characteristics of proteins such as folding, solubility,

resistance to proteases or thermal stability (Geyer et al., 2006; Morelle et al., 2006). The

potential role of the N-linked oligosaccharides of PgChP on intra- or extracellular

signalling could be essential for the antifungal activity of this protein.

The predicted mass of the translated nucleotidic sequence, according to the Compute

pI/Mw tool (Gasteiger et al., 2005), was 31,991 Da, which is close to the 30 to 31 kDa

determined for the six excreted isoforms of the protein by ESI-MS (Rodríguez-Martín et

al, 2009). The difference between the predicted mass and the determined weight can be

explained by the lack of the signal peptide (2,300 Da) in the excreted protein and the

presence of N-linked oligosaccharides. The predicted isoelectric point (Gasteiger et al.,

2005) was 4.8, which is close to the value of 5 determined by 2D-PAGE—IEF (Rodríguez-

Martín et al., 2009).



CAP80409) that differs from the precursor of PgChP in only four amino acids. However,

the production of the protein by this mould has not been reported.

In conclusion, the gene encoding the chitosanase PgChP with antifungal activity has been

sequenced. The putative amino acid sequence of PgChP shows two consensus sites for

glycosylations. The role of N-linked oligosaccharides from PgChP on antifungal activity

deserves further investigation.

RESULTADOS

127

ACKNOWLEDGEMENTS


06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were recipients of a FPI

grants from the Spanish Ministry of Education and Science. Authors would like to thank

Dr. Susan Liddell (Division of Animal Sciences) and Prof. Ian Connerton (Division of

Food Sciences) for their support during Andrea Rodriguez stay’s at Nottingham

University.

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Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium

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Shimosaka, M., Sato, K., Nishiwaki, N., Miyazawa, T., and Okazaki, M. Analysis of

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Manuscrito 6: Effect of different treatments

on chitosanase and antifungal activity of PgChP


RESULTADOS

131

Effect of different treatments on chitosanase and antifungal activity of

PgChP from Penicillium chrysogenum

Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Miguel A. Asensio*


Universidad, s/n. 10071- Cáceres, Spain.



ABSTRACT

The protein PgChP showed antifungal and chitosanase activities with potential application

in food industry as protective additive and production of chitosan oligomers, respectively.

Haemolytic assay was negative, essential characteristic for using it for human applications.

PgChP was resistant at heat treatments even higher than 60ºC. Different ions influenced on

chitosanase and antifungal activities of the protein; thus, Na+ and Mg2+ affected to the

chitosanase activity, whereas the antifungal activity was only affected for Mg2+ and Ca2+

and not for monovalent ions. Non-ionic detergents remained or improved the chitosanase

action, although decreased the antifungal activity. Ionic detergents inhibited both

antifungal and chitosanase activities. EGTA (0.1 mM), Triton X-100 (1%) and phosphate

(1 mM) increased the chitosanase activity of PgChP. The antifungal activity does not seem

to be related to chitosan degradation in sensitive moulds.

KEY WORDS: chitosan oligomer, chitosanase, antifungal activity, food preservative,

haemolytic activity.

INTRODUCTION

Natural antifungal proteins are a new source of clinically useful therapeutics. Antifungal

proteins have different action mechanisms. Some of them interfere on DNA, RNA, and

protein synthesis, or they can act on fungal walls in diverse ways. Some of them decrease

the ATP level (Cociancich et al.., 1993) or depolarize the membrane (Thevissen et al..,

1996). Other antifungal proteins interfere with essential elements of fungal walls, such as

�-1,3-glucan (Odds et al.., 2003), chitin (Yang and Gong, 2002; Von der Weid et al.,

2003), or chitosan (Davis and Eveleigh, 1984). These compounds can be even hydrolized

by some antifungal proteins like glucanases, chitinases, and chitosanases.

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132

The most useful antifungal proteins are those that act specifically against fungal

membranes because of their lesser toxic effects on mammalian cells (Groll et al., 1998).

However, none of these antifungal proteins is currently utilized because they have shown

to be cation-sensitive. Sensitivity to high-ionic-strength conditions may be a crucial point

in the application of antifungal proteins under physiological conditions, or in many food

products with high salt concentration. The protein PgChP was previously purified from the

strain P. chrysogenum AS51D for its ability to inhibit the growth of toxigenic moulds

(Acosta et al., 2009). Given that this mould was isolated from dry-cured ham, a substrate

with a high salt concentration, PgChP could withstand cations and have adequate

characteristics for the use in foods.

PgChP showed high homology with fungal chitosanases according to its amino acid

sequence (Rodríguez-Martín et al., 2009a and 2009b). Thus, PgChP could have

chitosanase as well as antifungal activity. Chitosanases can degrade a variety of chitosans

with different degrees of acetylation (Ohtakara et al., 1988; Osswald et al., 1993) to yield

chitosan oligomers, which have interesting biological properties, including antifungal and

antibacterial activity (Allan and Hadwiger, 1979; Hirano and Nagano, 1989), as food

preservatives (Rivas et al., 2000).

Chitosan is a polysaccharide found in fungal walls (Zhang et al., 2000; Kumiko et al.,

2002; Di Mario, 2008) and its degradation by chitosanases can be responsible for inhibiting

target moulds (Saito et al., 2009). Thus, the antifungal activity of PgChP could be related

to the chitosanase activity.

To design potential applications, the impact of environmental factors on PgChP activity

should be known. The aim of the present work was to study the influence of factors such as

temperature, different substrates, ions, and detergents on both antifungal and chitosanase

activities.


Microbial strain

The PgChP producing mould, P. chrysogenum AS51D, and the toxigenic mould

Penicillium commune Pc332 were isolated from dry-cured ham and belong to the fungal

collection of Food Hygiene (University of Extremadura, Spain).

RESULTADOS

133

PgChP purification

P. chrysogenum AS51D was grown at 25ºC for 15 days in malt extract broth made of

glucose 2% (w/v), malt extract 2% (w/v) and peptone 0.1% (w/v), pH 4.5. Then, the

medium was filtered though a 0.45 µm diameter pore size cellulose acetate membrane and

PgChP was purified using an ÄKTA fast protein liquid chromatography system

(Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009).

For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column

(Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH

4.5. Bound proteins were eluted with a gradient of 0.02 M sodium acetate, 1 M NaCl

buffer, pH 4.5: 0% to 25% in 15 column volumes (CV), increase to 100% in 2 CV, and

finally keeping 100% for 15 CV. Eluted proteins were detected at 214 nm. The fraction

that showed antifungal activity in previous studies (Acosta et al., 2009) was purified using

a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia

Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7, at a

constant flow rate of 2.5 ml/min. Data collection and processing were done using the

UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction

containing PgChP was desalted and concentrated with YM-10 microcon centrifugal filter

units (Millipore Iberica S.A.U., Madrid, Spain) and kept at -20ºC.

Protein was quantified by Johnson method according to Córdoba (1990). An aliquot of the

purified protein (100µl) was dried in a heat cabinet at 100 ºC. The sample was digested by

adding 200 µl of sulfuric acid in a sand bath at 120 ºC. Then, 4.8 ml of distilled water, 3 ml

of 4N NaOH and 2 ml of Nessler reactive made of KI 0.4% (w/v), 0.4% HgI2 (w/v) and

0.175% (w/v) arabic gum were added. After 10 min at room temperature the absorbance

was measured at 490 nm in a spectrophotometer. A calibration curve was obtained with

different concentrations of ammonium sulfate.

Sodium dodecyl sulphate-polyacrylamide gel eletrophoresis (SDS-PAGE)

SDS-PAGE was performed as described Laemmli (1970). The protein PgChP was

separated on a 4% (v/v) polyacrylamide stacking and 8% (v/v) polyacrylamide resolving

gels. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford,

U.S.A.). The molecular weight was determined by comparison of its electrophoretic

mobility with proteins of the molecular mass marker (66-6.5 kDa) from Sigma (Sigma-

Aldrich Química S.A., Madrid, Spain).

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Haemolytic activity assay

The haemolytic activity of the protein was determined using sheep defibrinated blood

(Oxoid). The erythrocytes were isolated by centrifugation (1000 ×g for 6 min at room

temperature) and washed three times with 50 mM sodium phosphate, 0.15 M NaCl buffer,

pH 7. The cell concentration in the stock suspension was adjusted to 1×109 cells/ml. A

volume of 50 µl of cells suspension was added to 200 µl of sterile concentrated protein (10

µg/ml) and incubated at 37ºC. After 0, 8, and 24 h, the samples were placed on ice,

immediately centrifuged at 4ºC, and the supernatant was carefully collected. The

haemolysis was determined by measuring absorbance of the supernatant at 570 nm

(Moerman et al., 2002) and 541 nm (Lai et al., 2005). Controls for 0% and 100%

haemolysis were obtained suspending sheep red blood cells in the buffer or distilled water,

respectively. The haemolysis percentage was calculated using the following equation:

Percentage haemolysis of supernatants = [(Abs in the peptide solution − Abs in buffer)/Abs

in water solution] × 100.

Three independent assays were conducted with triplicated readings for each one.

Evaluation of chitinase and chitosanase activities

Chitinase and chitosanase activities were determined with a modification of the 3,5-

dinitrosalicylic acid method (Su et al., 2006) by measuring the reducing sugars liberated

during the hydrolysis of chitin or chitosan. One ml of the 1% (w/v) chitin or chitosan

(Sigma-Aldrich) in 1% (v/v) acetic acid was added to 1 ml of the PgChP solution

(10µg/ml) and incubated for 2-4 h at 37ºC. Then, to detect the hydrolisis reaction, 2 ml of

dinitrosalicylic acid reagent, made of 1% (w/v) 3,5-dinitrosalicylic acid (Sigma-Aldrich),

0.05% (w/v) sodium sulfite, and 1% (w/v) phenol were added and the sample was

incubated at 100ºC for 45 min. Finally, 1 ml of 40% (w/v) potassium sodium tartrate was

added and the solution was centrifuged. The supernatant was measured at 520 and 575 nm.

To quantify the reducing sugars liberated, a calibration curve was obtained with different

concentrations of D-(+)-Glucosamine hydrochloride (Sigma-Aldrich). A chitinase from

Streptomyces griseus (Sigma-Aldrich) with both chitinase and chitosanase activities was

used as positive control.

Preparation of colloidal chitin: The colloidal chitin was obtained following the method of

Roberts and Selitrennikott with minor modifications (Harighi et al., 2007). Ten grams of

RESULTADOS

135

chitin powder were added slowly into 100 ml of H3PO4 (85%) at 25ºC under vigorous

shaking for 2 h. This solution was carefully added over 1 liter of absolute ethanol at -20ºC.

The mix was shaken for 30 min and stored until use. Ten ml of suspension were

centrifuged, the pellet was washed three times with 50 mM sodium phosphate buffer pH 7,

and resuspended in distilled water. The final concentration of colloidal chitin was around

1%.

Chitinase activity assay by gel electrophoresis

The chitinase activity assay was carried out as described by Trudel and Asselin (1989).

PAGE-SDS was performed in 4% (v/v) polyacrylamide stacking gel and 8% (v/v)

separating polyacrylamide gradient gel containing 0.01% (w/v) glycol chitin and 0.1%

(w/v) SDS. Samples were boiled for 5 min with 20% (v/v) glycerol, 0.025% (w/v)

bromophenol blue, 2% (w/v) SDS and 5% (v/v) �-mercaptoethanol in 62.5 mM Tris-HCl

(pH 6.8). The gel was run at 100 V for 1 h.

After electrophoresis, the gel was incubated for 2 h at 37 ºC under continuous shaking in

100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100. Then, the gel

was stained with a 0.01% (w/v) calcofluor white M2R (Sigma-Aldrich) in 500 mM Tris-

HCl (pH 8.9). After 5 min, this solution was removed and the gel was incubated in distilled

water at room temperature for about 1 h. Lytic zones were visualized in the UV

transilluminator Syngene G-Box (Synoptics, Fredericks, USA). The gel was photographed

and analyzed with the GeneSnap software (Synoptics). An additional gel was stained only

with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford, U.S.A.). The chitinase

from S. griseus (Sigma) was used as positive control. The molecular mass marker used was

66-6.5 kDa (Sigma).

Glycol chitin synthesis: 0.2 g glycol chitosan was dissolved in 4 ml of 10% acetic acid and

the mixture was shaken overnight at 22ºC. Methanol (18 ml) was slowly added and the

solution was filtered through 0.45 µm pore size cellulose acetate membrane filters. Acetic

anhydride (0.3 ml) was added and kept for 30 min. The resulting gel was cut into small

pieces and after adding 1 vol of methanol was shaken for 4 min. This suspension was

centrifuged at 27000 ×g at 4ºC for 15 min and the pellet was resuspended in 20 ml of

0.02% (w/v) sodium azide. The final concentration of the glycol chitin was 1% (w/v).

MANUSCRITO 6

136

Evaluation of Antifungal activity

Quantitative assay for fungal growth inhibition was performed following the protocol

developed by Broekaert et al. (1990) with some modifications (Acosta et al., 2009). P.

commune Pc332 (Núñez et al., 1996) was used as reference strain. To perform the

inhibition test in microtitter plates, 100 µl of the fraction obtained by gel filtration

chromatography were dispensed in triplicate wells. Each well was loaded with 100 µl of

concentrated malt extract broth (4% malt extract, 4% glucose and 0.2% peptone, pH 4.5)

inoculated with the reference strain to c.a. 106 spores/ml. The plates were incubated for up

to 72 h at 25 ºC. Fungal growth was determined by measuring the optical density variation

at 595 nm.

Effect of heat-treatments

The study of the protein stability after heat treatment was carried out according to Okkers

et al. (1999). Aliquots of PgChP protein (10 µg/ml) were exposed to different

combinations of temperatures (60, 80, and 100 ºC) and times (10, 20, and 30 min), as well

as to sterilization at 121 ºC for 15 min. After the heat-treatment, the samples were cooled

on ice. Then, antifungal and chitosanase activities were evaluated as described above.

Influence of different chemicals on chitosanase and antifungal activity

To study the influence of some chemicals on the protein, the activity of PgChP (10 µg /ml)

was tested with different compounds: NaCl, CaCl2, KCl, or MgCl2 (0.1, 1, 10, and 100

mM); EDTA or EGTA at 0.01, 0.1, and 1 mM; 0.5% and 1% of Triton X-100, tween-20,

tween-80, SDS, or deoxycholic acid; NaH2PO4 at 0, 0.1, 1, 10, and 100 mM.

Statistical analysis

Data were statistically analyzed using SPSS software package (version 13.0. SPSS Inc.,

Chicago, USA) by a variance analysis (one way ANOVA) and the means were separated

by the Tukey honest significant difference test.

RESULTADOS

137

RESULTS

Haemolytic activity

To study the haemolytic activity of PgChP on mammalian cells, the effect of the protein

was examined on sheep red blood cells. The protein PgChP did not show haemolytic

activity, being the value obtained of 0%, even after 24 h.

Chitosanase and chitinase activities

The protein PgChP showed chitosanase activity, but no chitinase activity was found by the

3,5-dinitrosalicylic acid method (Figure 1).

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Figure 1. Chitinase and chitosanase activities of PgChP expressed as liberated D-glucosamine after

4 h. Asterisks indicate statistically significant differences to the negative control without protein

(***p<0.001). Chitinase from S. griseus (50 µg/ml) was used as positive control.

To further evaluate the chitinase activity, the hydrolysis of chitin was tested by

electrophoresis in a glycol chitin gel. No activity was observed loading the polyacrilamide

gel with 18 µg of PgChP (Figure 2). Lytic zones by chitinase from S. griseus, used as

positive control, were observed as black bands after staining with calcofluor white M2R.

MANUSCRITO 6

138

Protein stability to different temperatures

PgChP protein showed a remarkable stability at high temperatures. The chitosanase

activity did not show a significant reduction after heat treatments even at 60ºC for 30 min

(Figure 3).

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Figure 3. Effect of heat-treatment on PgChP chitosanase activity expressed as mean and standard

deviation of liberated glucosamine after 2 h. Asterisks indicate statistically significant differences

(p<0.05) to the positive control without heat-treatment (C+).

Figure 2. Chitinase activity of PgChP evaluated by gel electrophoresis. SDS-PAGE gels

with glycol chitin were stained with Imperial Protein Stain (A) and Calcofluor White M2R

(B). M: protein marker, Q: Chitinase from S. griseus (100 µg).

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RESULTADOS

139

The antifungal activity seems to withstand even treatments of 80ºC up to 20 min (Figure

4), whereas higher temperatures inactivated PgChP antifungal activity allowing an

unrestricted growth of the sensitive mould.

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Figure 4. Antifungal activity of heat-treated PgChP expressed as mean and standard deviation of P.

commune Pc332 growth after 48 h. Asterisks indicate statistically significant differences (p<0.05)

to the positive control with non heat-treated PgChP. C+: positive control with non heat-treated

PgChP; C-: negative control without PgChP.

Effect of ions on PgChP chitosanase and antifungal activities

PgChP chitosanase activity was not affected by the presence of up to 100 mM CaCl2 or

KCl ions whereas it decreased at the highest NaCl or MgCl2 concentration (Figure 5).

MANUSCRITO 6

140

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Figure 5. Effect of monovalent (A) and divalent (B) ions on the chitosanase activity of PgChP

expressed as mean and standard deviation of liberated glucosamine after 3 h. Asterisks indicate

statistically significant differences to the positive control without ions (closed bars) (**p<0.01;

***p<0.001).

On the other hand, the inhibition of the reference mould was unaffected by monovalent

ions, K+ and Na+, but significantly decreased with high concentrations of divalent ions

(Figure 6).

RESULTADOS

141

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Figure 6. Effect of monovalent (A) and divalent (B) ions on antifungal activity of PgChP against

P. commune Pc332 expressed as mean and standard deviation of optical density after 72 h.

Asterisks indicate statistically significant differences to the negative control (C-) without PgChP (*

p<0.05; ** p<0.01; *** p<0.001).

Effect of cation chelators

Activity of PgChP was also assayed in presence of EDTA and EGTA. The chitosanase

activity was strongly affected by high levels of chelators. Thus, 1 mM EDTA inhibited

chitosanase activity, whilst even low levels of EGTA increased chitosan hydrolysis by

PgChP (Table 1).

MANUSCRITO 6

142

Table 1. Effect of EDTA and EGTA on chitosanase activity of PgChP after 2 h incubation.

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a Results are expressed as mean ± SD of liberated glucosamine. Asterisks indicate statistically

significant differences to the positive control without chelators (*p<0.05; ***p<0.001).

The antifungal activity of PgChP was observed at the lowest EDTA concentration (0.01

mM) but not at 0.1 mM (Table 2). The highest level of EDTA tested inhibited growth of

the reference mould, and no further effect on the antifungal activity could be observed.

EGTA did not seem to inhibit growth of the reference mould even at the highest

concentration tested. No effect of EGTA was observed on the antifungal activity against P.

commune Pc332, irrespectively of the concentration used (Table 2).

Table 2. Effect of EDTA and EGTA on the antifungal activity of PgChP against P. commune

Pc332 after 48 h incubation.

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differences to the negative control without PgChP (C-) (*p<0.05; **p<0.01; ***p<0.001).

RESULTADOS

143

Resistance to protein-denaturing detergents

Detergents displayed different effects on the chitosanase activity of PgChP (Figure 7).

Non-ionic detergents showed no inhibition on the activity. In fact, tween 20, tween 80, and

Triton X-100 even increased the chitosanase activity. In contrast, the ionic detergents

sodium dodecil sulphate (SDS) and deoxycholic acid partially or completely inhibited the

chitosanase activity, respectively, for the 2 h incubation time.

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standard deviation of liberated glucosamine after 2 h. Asterisks indicate statistically significant

differences to the positive control without detergents (*p<0.05; ***p<0.001).

On the other hand, PgChP showed no antifungal activity on P. commune Pc332 with 0.5 or

1% (w/v) of the tested detergents.

MANUSCRITO 6

144

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expressed as mean and standard deviation of optical density after 72 h. Asterisks indicate

statistically significant differences to the negative control (C-) without PgChP (*** p<0.001).

Effect of sodium phosphate on the PgChP activity

The chitosanase activity decreased at the highest concentration of sodium phosphate tested,

whereas levels below 100 mM favoured this activity (Table 3).

Table 3. Influence of phosphate on the chitosanase activity of PgChP after 2 h of incubation.

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a Results are expressed as mean ± SD of liberated glucosamine. Asterisks indicate statistically

significant differences to the positive control without phosphate (** p<0.01; *** p<0.001).

Phosphate also seems to affect the antifungal activity of PgChP, but the uneven growth of

the reference mould in negative controls did not allow to obtain more consistent results

(Table 4).

RESULTADOS

145

Table 4. Influence of phosphate on the antifungal activity of PgChP against P. commune Pc332

after 72 h.

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differences to the negative control without PgChP (** p<0.01; *** p<0.001).

DISCUSSION

As it has been shown in a previous work, the protein PgChP showed antifungal activity

against P. commune Pc332 (Acosta et al., 2009), a toxigenic mould present in foods (Sosa

et al., 2002). PgChP was obtained by ion exchange chromatography looking for cationic

proteins (Acosta et al., 2009), because the cationic character in proteins is related to a

higher antifungal activity, but to a lower toxicity for mammalian cells (Hong et al., 2001).

Given that PgChP shows an isoelectric point close to 5 (Rodríguez-Martín et al., 2009a),

the protein is not cationic under physiological conditions and its potential as toxic for

humans has to be assessed. The haemolysis assay to test for cytolytic activity is generally

accepted as proof that the protein can be regarded as safe (Van’t Hof et al., 2001). PgChP

did not show any haemolytic activity even though in depth tests are needed to confirm the

safety of this protein.

The amino acid sequence of PgChP revealed a high homology to fungal chitosanases

(Rodríguez-Martín et al., 2009a, 2009b). Thus, the chitosanase activity of PgChP was

evaluated. As it is shown in figure 1, the protein showed a significant chitosanase activity.

Some microbial chitosanases, such us the chitosanase II from Acinetobacter sp. strain

CHB101, hydrolyze colloidal chitin and glycol chitin besides chitosan (Shimosaka et al.,

1995). However, PgChP did not show activity on either of these substrates, which agrees

with the fact that most chitosanases do not have chitinase activity (Su et al., 2006;

Fukamizo and Brzezinski, 1997).

Fungal chitosanases differ in their thermostability. There are chitosanases such as Csn2

from Gongronella sp. JG (Wang et al., 2008) that resist 50 and 55ºC for 30 and 11 min,

respectively. Bacterial chitosanases have shown stability within a similar or lower range of

MANUSCRITO 6

146

temperature, such as those from Acinetobacter sp. C-17 (Zhu et al., 2003) and

Sphingomonas sp. CJ.5, that are stable at 30-40ºC and 45ºC for one hour, respectively (Zhu

et al., 2007). Given that PgChP kept chitosanase activity after heating at 60ºC for 30 min

(Figure 3), it can be regarded as more stable than other chitosanases.

On the other hand, the antifungal activity of PgChP was more heat-resistant than

chitosanase activity. PgChP antifungal activity resisted even at 80 ºC for up to 20 min. The

fact that chitosanase and antifungal activities of PgChP differ in thermostability indicates

that chitosan-hydrolysis is not essential for the antifungal activity, as it will be discussed

later.

The presence of K+ and Ca2+ did not affect the chitosanase activity of PgChP at the highest

tested concentration, but 100 mM Mg2+ and Na+ inhibited that activity. However, K+ and

Ca2+ activate several fungal chitosanases (Alfonso et al., 1992; Fenton and Eveleigh,

1981). As a consequence, metal ions can have different impact on fungal chitosanases.

This effect can be related to the influence of different concentrations of particular ions on

the structure of chitosanases (Wang et al., 2008). Regarding the antifungal activity of

PgChP, over 1 mM Mg2+ or Ca2+ reduced the inhibition on P. commune, whereas

monovalent ions Na+ or K+ did not affect the antifungal activity (Figure 6). The fact that

divalent ions repressed the antifungal activity could mean that these ions compete with

PgChP for potential target sites in the fungal wall, as it has been described for metal ions

on other antifungal proteins (Broekaert et al., 1992; Kaiserer et al., 2003; Theis et al.,

2003). In addition, the divergent effect of Na+ and Ca2+ on PgChP chitosan hydrolysis and

fungal inhibition supports that different mechanisms of action are responsible for these

activities.

The activity of proteins can also be influenced by low concentrations of metal ions. The

addition 1 mM of EDTA, a chelator of Mg2+ and Ca2+ (Choucair et al., 2006), decreased

the chitosanase activity. Then, the absence of Mg2+ seems to inhibit the chitosanase activity

of PgChP. In contrast, EGTA inhibited neither chitosanase nor antifungal activity of

PgChP, and even notably increased the chitosanase activity (Table 1). EGTA is a quite

specific chelator of Ca2+ (Choucair et al., 2006). Thus, the chitosanase activity of PgChP

was not affected by the absence of this ion.

On the contrary, the antifungal activity was not affected by EGTA or low levels of EDTA

(Table 2), whereas high levels of EDTA strongly limited the development of the reference

RESULTADOS

147

mould. Once more, the differences on chitosanase and antifungal activities of PgChP are

evidenced, due to the impact of low levels of metal ions.

Ionic and non-ionic detergents had a distinct effect on PgChP activities. Non-ionic

detergents (tween 20, tween 80, and Triton X-100) led to an increase of chitosanase

activity (Figure 7), probably due to an improved protein-substrate interaction. However, no

clear effect of these detergents could be observed on the antifungal activity, due to the low

growth of P. commune Pc332 even when added at 0.5%.

The ionic detergents SDS and deoxycholic acid caused loss of both chitosanase and

antifungal activities. The effect of these detergents can be attributed to protein denaturation

that prevents PgChP from being active.

Finally, sodium phosphate increased the chitosanase activity of PgChP, except at the

highest concentration (100 mM) which caused loss of activity. These results are somehow

similar to those obtained with identical levels of NaCl. Thus, the main impact of sodium

phosphate on this activity can be attributed to Na+ concentration. The evaluation of

phosphate effect on the antifungal activity was hampered by an uneven growth of Pc332 in

control wells without PgChP. As it can be observed in table 4, 10 mM phosphate PgChP

exerted a significant inhibition (p<0.01) on P. commune, but this was not observed at lower

concentrations. The poorer growth reached by P. commune in the negative controls can

mask statistically significant differences due to the antifungal activity.

As it has been evidenced mainly by the residual activity after heat treatments and under

high Na+ or Ca+ concentrations, antifungal and chitosanase activities of PgChP are

independent from each other. This is in contrast to the antifungal activity of the protein

CtoA from Amycolatopsis sp. that has been attributed to the hydrolysis of chitosan in the

cell wall of the sensitive Rhizopus oryzae (Saito et al., 2009). In addition, it is unlike that

the antifungal activity of PgChP is related to the degradation of chitosan in the fungal wall

because chitosan has not been described in P. commune.

Applications of PgChP for chitosan-hydrolysis could take advantage of the thermal

stability to 60ºC and the increased activity of the protein by adding EGTA (0.1 mM),

Triton X-100 (1%) or phosphate (10 mM). Nontheless, the conditions of pH and

temperature for an optimum chitosanase activity should be determined.

The antifungal activity of the PgChP protein was not affected by high temperatures and

monovalent cations, indicating that PgChP have potential applications as a preservative in

MANUSCRITO 6

148

the food industry, particularly on substrates with a high concentration of salts. Thus, the

addition of PgChP could prevent the growth of unwanted moulds in dry-cured meat

products, where Na+ and K+ are abundant. Even though a complete study about the safety

of PgChP is necessary, the information here reported is promising for a potential use of this

protein as food preservative.

ACKNOWLEDGEMENTS


06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of a

FPI grant from the Spanish Ministry of Education and Science.

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V. DISCUSIÓN

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V. DISCUSIÓN

Las proteínas PgAFP y PgChP, purificadas a partir de dos cepas de Penicillium

chrysogenum aisladas de jamón curado, mostraron actividad antifúngica en estudios

previos. Debido a la potencialidad que esta actividad presenta para su aplicación en el

control de mohos indeseables en alimentos se planteó la caracterización de dichas

proteínas.

V. 1. CARACTERIZACIÓN BIOQUÍMICA

La masa molecular de la proteína PgAFP se había estimado en torno a 9 kDa

mediante SDS-PAGE. Sin embargo, el análisis de la proteína purificada mediante

espectrometría de masas SELDI-TOF (Acosta y col., 2009b) y ESI-MS (Manuscrito 1)

reveló un peso molecular de únicamente 6,5 kDa (Figura V.1).

Esta discrepancia entre los resultados de las dos técnicas podría deberse a la

existencia de modificaciones post-traduccionales en la proteína (Pitt-Rivers y Ambesi

Impiombato, 1968; Tozzini y col., 1994; Murayama y col., 2003). Así se han descrito

migraciones anómalas de proteínas glicosiladas en SDS-PAGE atribuidas a una menor

capacidad de las mismas para unirse al SDS (Nelson, 1971; Russ y Poláková, 1973; Bagger

y col., 2007). PgAFP mostró además una reacción positiva a la tinción con ácido

periódico-Schiff (Acosta y col., 2009b), el cual oxida los grupos 1,2-glicol presentes en los

polisacáridos a aldehídos, que a su vez reaccionan con la fucsina para formar bandas de

color magenta o rosado (Zacharius y col., 1969). Por lo tanto, este resultado es indicativo

de la presencia de cadenas de glicano unidas a la proteína. Un posterior análisis de la

proteína mediante espectrometría de masas MALDI-TOF reveló la presencia de picos

Figura V.1. Peso molecular de PgAFP en SDS-PAGE (A) y análisis por SELDI-TOF (B;

Acosta y col., 2009b).

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DISCUSIÓN

154

atípicos de 215 Da que no pudieron interpretarse como secuencias de aminoácidos, lo que

apoyó la hipótesis de que PgAFP tuviera algún tipo de glicosilación unida a su núcleo

proteico (Acosta, 2006). No obstante en el presente estudio, el tratamiento con una amplia

variedad de deglicosidasas no reveló ningún indicio de glicosilaciones, hecho que fue

confirmado posteriormente con la secuencia deducida a partir del gen que codifica dicha

proteína (Manuscritos 1 y 2). En consecuencia, la migración anómala mostrada por PgAFP

en los geles de poliacrilamida podría ser explicada por su alto punto isoeléctrico, estimado

en 9,2 (Acosta, 2006), que puede originar repulsiones electrostáticas que dificulten la

unión con el SDS impidiendo una correcta migración en los geles (Mondstadt y Halldorf.,

1990).

De manera similar, la proteína PgChP también mostró diferencias en el peso

molecular obtenido mediante SDS-PAGE y espectrometría de masas, aunque en esta

proteína sí pudo confirmarse la existencia de glicosilaciones (Manuscrito 4). Durante la

purificación de la proteína se procedió a su concentración, lo que permitió visualizar dos

bandas de 37 y 40 kDa en gel de poliacrilamida (Manuscrito 4, figura 1), la segunda de

ellas no detectada anteriormente (Acosta y col., 2009a). Después de tratamientos de

deglicosilación con PNGasa F se observó una única banda de 37 kDa en el gel. Estos

resultados indican que la cadena proteica posee esta masa molecular y posteriormente es

glicosilada, formándose una molécula de 40 kDa. Adicionalmente, la secuenciación

aminoacídica de ambas proteínas de 37 y 40 kDa reveló secuencias de aminoácidos

idénticas (Tabla 3, manuscrito 4), ratificando que las dos bandas observadas en los geles

correspondían a dos isoformas de la misma proteína, una de ellas glicosilada.

Posteriormente, un análisis más exhaustivo mediante espectrometría de masas con

fuente de ionización por electrospray reveló la existencia de glicosilaciones en PgChP, ya

que se detectaron seis isoformas de entre 30 y 31 kDa (Figura V.2) cuyo peso molecular

difería en múltiplos de 162 Da, masa que se corresponde con un residuo de galactosa o

manosa.

DISCUSIÓN

155

La glicosilación de la proteína se confirmó posteriormente mediante el análisis de la

secuencia de aminoácidos que se deduce del gen que codifica PgChP (Manuscrito 5). Así,

la diferencia entre el peso molecular de la proteína deducida (29691 Da) y el valor

experimental de cada isoforma (30285, 30447, 30610, 30772, 30934 y 31098 Da),

corresponde a la parte glicídica de cada una de ellas, lo cual supone un nivel de

glicosilación en PgChP que va desde un 1,6 a un 4,5 % dependiendo de la isoforma.

Adicionalmente, el análisis tanto de los péptidos obtenidos tras la digestión con tripsina y

quimotripsina, como de la secuencia de aminoácidos deducida del gen que codifica PgChP,

reveló la presencia de dos secuencias consenso de glicosilación (Morelle y col., 2006) en

esta proteína: Asn-Phe-Thr (aminoácidos 230-232) y Asn-Asp-Cys (aminoácidos 288-

290).

La glicosilación representa la forma más común de las modificaciones post-

traduccionales en proteínas tanto de eucariotas como de procariotas, donde participan en

numerosos procesos biológicos, tales como señalización inter- e intracelular, regulación de

proteínas o reconocimiento celular. Los glicanos pueden determinar la localización,

actividad y función de las proteínas que los contienen, así como influir en las

características fisicoquímicas de las mismas, tales como plegamiento, solubilidad,

resistencia a proteasas o aumento de la estabilidad a altas temperaturas (Hildegard y Geyer,

2006; Morelle y col., 2006). De hecho estos grupos glicídicos parecen estar implicados en

la actividad antifúngica de algunas proteínas purificadas a partir de plantas (Islam y col.,

2002; Deepak y col., 2003, Yamunarani y col., 2005; Kuo y col., 2008).

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Figura V.2. Peso molecular de las seis isoformas de PgChP determinado por ESI-MS.

DISCUSIÓN

156

Profundizando en el estudio de las secuencias de aminoácidos deducidas a partir de

los genes que codifican la producción de las proteínas PgChP y PgAFP, el análisis con el

programa SignalP (Nielsen y col., 1997a) permitió predecir la existencia de péptidos señal

o presecuencias en ambas proteínas. Estas secuencias comprenden los primeros 21

(MMTYSRLIPFALFALFGTGLA) y 18 (MQITSIAIVFFAAMGAVA) aminoácidos del

extremo N-terminal de PgChP y PgAFP, respectivamente (Manuscritos 2 y 4).

En el caso de PgAFP pudo confirmarse además la presencia de una prosecuencia

comprendida por los aminoácidos 19 a 34. Este fragmento, que difiere del péptido señal,

no está presente en la proteína madura, por lo que no pudo obtenerse mediante la

secuenciación aminoacídica de la proteína purificada a partir del medio de cultivo. Se han

descrito prosecuencias similares en proteínas con actividad antimicrobiana producidas

tanto por mohos (Oka y col., 1990; Wnendt y col., 1994; Marx y col., 1995), como por

levaduras (Tao y col., 1990; Schmitt y Breinig, 2002) y bacterias (Bormann y col., 1999),

que mantienen inactiva a la proteína en el interior de la célula. De hecho, es necesaria la

eliminación de estos fragmentos durante el proceso de secreción al medio para que la

proteína adopte una correcta estructura terciaria y adquiera su actividad (Eder y Fersht,

1995).

El análisis de las secuencias de aminoácidos deducidas permitió además estimar

tanto la masa como el pI de ambas proteínas (Gasteiger y col., 2005). Así, las masas

moleculares estimadas de PgChP y PgAFP una vez eliminados los respectivos péptidos

señal y la preprosecuencia fueron de 29691 y 6500 Da, respectivamente. Los pIs estimados

para PgChP (pH 4,8) y PgAFP (pH 8,8) fueron muy próximos a los de 5,0 y 9,2

respectivamente, determinados por electroforesis en gel de dos dimensiones (Manuscrito 4,

figura 5) y mediante electroforesis capilar de isoelectroenfoque (Acosta y col., 2009b).

Numerosos autores han relacionado el carácter catiónico con la actividad antifúngica

(Broekaert y col., 1992; Nielsen y col., 1997b; Koo y col., 1998; Hong y col., 2001), ya

que la presencia de aminoácidos de carga neta positiva sugiere la capacidad de

interaccionar con los fosfolípidos aniónicos de membrana de mohos (Sitaram y Nagaraj,

1999) pudiendo provocar alteraciones en la permeabilidad de la misma. Por lo tanto, el

punto isoeléctrico de ambas proteínas puede tener una gran relación con la actividad de las

mismas, fundamentalmente en el caso de PgChP, ya que presentan una carga neta negativa

a pH fisiológico. No obstante, la posible implicación de la carga en la actividad de ambas

proteínas se comentará más adelante.

DISCUSIÓN

157

V. 2. CARACTERIZACIÓN GENÉTICA

Los genes que codifican la producción de PgAFP y de PgChP fueron secuenciados

mediante RACE-PCR. Los genes pgafp y pgchp, compuestos por 276 y 912 nucleótidos,

codifican los precursores de 92 y 304 aminoácidos, respectivamente (Tabla V.1). En

ambos casos el marco de lectura abierto (ORF) está interrumpido por pequeños intrones de

63 y 62 pb en pgafp, y de 60, 53, 59 y 59 pb en pgchp, típicos de genes fúngicos (Gurr y

col., 1987). Todos los intrones incluyen en los extremos 5’ y 3’ las secuencias consenso de

ensamblamiento o “consensus splice sequences” (Manuscrito 2, figura 5; Manuscrito 5,

figura 4), y muchos de ellos además contienen la secuencia interna CTRAC, siendo todas

estas secuencias típicas de genes fúngicos (Wiesner y col., 1988).

Tabla V.1. Características de los genes pgafp y pgchp.

Uno de los factores más importantes para emplear una proteína a nivel industrial es

el bajo coste para producirla. Para identificar las condiciones de cultivo ideales y los

factores que estimulan su producción es necesario conocer los mecanismos que regulan la

expresión de los genes que la codifican. En este sentido, el estudio de la región promotora

de los genes proporciona valiosa información sobre las condiciones de expresión de las

proteínas que codifican. Así, la caracterización del promotor del gen que codifica para la

proteína PAF de P. chrysogenum permitió confirmar la presencia de cuatro sitios de unión

a CreA de represión de catabolitos de carbono y dos sitios de unión a AreA de represión de

metabolitos de nitrógeno (Marx, 2004). En este tipo de regulación, la transcripción del gen

se inhibe en presencia ciertos hidratos de carbono, fundamentalmente glucosa y fructosa

(Marx y col., 1995; Vollebregt y col., 1994), y algunos compuestos nitrogenados pueden

tanto inhibir como intensificar la producción, como es el caso de glutamina y amonio

respectivamente (Marx y col., 1995). Por otra parte, la expresión de la proteína AFP

producida por A. giganteus está regulada por otro tipo de factores ambientales como el pH

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DISCUSIÓN

158

y la presencia de fosfatos (Meyer, 2008). De hecho, el gen afp posee dos secuencias

consenso para un factor de regulación por pH (Tilburn y col., 1995) que hacen que la

producción de esta proteína se incremente cuando el moho productor se incuba en medios

alcalinos (Meyer y Stahl, 2002). La presencia de fosfatos por el contrario provoca una gran

disminución de la producción (Meyer y Stahl, 2002).

Debido a la influencia que numerosos factores ambientales pueden ejercer sobre la

expresión de PgAFP y PgChP, se hace necesario plantear nuevas investigaciones

encaminadas a la obtención de la región no codificante de pgafp y pgchp y al estudio de los

elementos de regulación presentes en los promotores, lo que permitirá optimizar las

condiciones de obtención de ambas proteínas. Por otra parte, ya se están llevando a cabo

diferentes ensayos para la expresión de ambos genes en microorganismos con mayor

velocidad de crecimiento como Escherichia coli o Pichia pastoris lo que facilitará la

obtención rápida de grandes cantidades de proteína, y de esta forma se posibilitará la

evaluación para una posible aplicación a nivel industrial. La clonación en E. coli permitirá,

en el caso de PgChP, la expresión de la proteína en un microorganismo procariota

obteniéndose una proteína recombinante sin glicosilaciones. Esta PgChP recombinante

puede resultar de gran interés para evaluar el papel que desempeñan las glicosilaciones en

la proteína y si este tipo de modificaciones post-traduccionales son necesarias para su

actividad antifúngica.

V. 3. COMPARACIÓN DE LA SECUENCIAS AMINOACÍDICAS DE PgAFP Y PgChP

CON OTRAS PROTEÍNAS

La caracterización a nivel bioquímico de PgChP y PgAFP reveló la existencia de

grandes diferencias entre ellas, aún tratándose ambas de proteínas con actividad

antifúngica producidas por P. chrysogenum. Así, en el caso de PgChP se trata de una

glicoproteína aniónica de unos 30 kDa que en principio muestra escasas características

comunes a PgAFP, de sólo 6,5 kDa y marcado carácter catiónico. El análisis de las

secuencias tanto aminoacídica como genética y su posterior comparación en bases de datos

permitió corroborar la existencia de tales diferencias. De hecho PgChP y PgAFP mostraron

semejanzas con diversas proteínas producidas por mohos con actividad quitosanasa o

antifúngica respectivamente.

PgChP reveló homologías tanto con quitosanasas fúngicas descritas como con

secuencias deducidas del genoma de distintos mohos, aunque la mayor similitud se

DISCUSIÓN

159

encontró entre PgChP y siete quitosanasas fúngicas con los siguientes números de

adquisición en Genbank: XP_754126 (Aspergillus fumigatus), XM_001262949 (N.

fischeri), XM_001390407 (Aspergillus niger), BAD08218 (Aspergillus oryzae-1),

XP_001824257 (A. oryzae-2), XM_001209034 (Aspergillus terreus) y XP_001274664

(Aspergillus clavatus). PgChP presentó cerca del 60% de homología con las siete

proteínas, siendo la más parecida la quitosanasa CSN de A. fumigatus.

Las quitosanasas (E.C. 3.2.1.132) son glicosil hidrolasas (glicosidasas) que cortan

enlaces glicosídicos del quitosán, y son producidas por virus, bacterias y hongos (Wang y

col., 2008a, Wang y col., 2008b). En función de sus secuencias aminoacídicas se han

clasificado en cinco familias: GH-5, GH-8, GH-46, GH-75 y GH-80. Las quitosanasas

fúngicas están incluidas en la familia GH-75 (Cheng y col., 2005), compuesta

principalmente por glicosidasas producidas por mohos de los géneros Aspergillus y

Fusarium (Shimosaka y col., 1996; Cantarel y col., 2008).

Como era de esperar, la secuencia aminoacídica deducida del gen pgchp presentó

numerosas regiones comunes con las siete proteínas que mostraron mayor homología

(Manuscrito 5, figura 5), incluyendo tres regiones conservadas en quitosanasas muy

relacionadas con su actividad y dos aminoácidos, glutámico y aspártico, esenciales para la

función catalítica de este tipo de enzimas, tal como se ha descrito para la quitosanasa de F.

solani (Shimosaka y col., 2005). De la misma manera, los diez residuos de cisteína

presentes en PgChP están conservados en las siete quitosanasas del alineamiento, y

probablemente confieren estabilidad mediante la formación de enlaces disulfuro (Nakaya y

col., 1990; Marx y col., 1995; Lee y col., 1999).

Por otra parte, en la familia GH-75 existen varias hidrolasas producidas por

bacterias del género Streptomyces. PgChP mostró una homología del 25% con

quitosanasas deducidas de los genes de S. griseus y S. avermitilis. Dado que se ha descrito

que las quitosanasas bacterianas y fúngicas tienen un origen evolutivo diferente (Zhang y

col., 2000; Shimosaka y col., 2005), esa similitud se debería a la presencia de una serie de

aminoácidos de gran importancia para la estructura y la actividad catalítica del enzima

(Monzingo y col., 1996). De hecho los aminoácidos glutámico y aspártico, esenciales para

la actividad de las quitosanasas fúngicas (Shimosaka y col., 2005), están presentes también

en estas dos proteínas bacterianas (Figura V.3). Por el contrario, las tres secuencias

altamente conservadas en quitosanasas fúngicas no parecen tener relación con las

homólogas en las secuencias de las proteínas bacterianas (Figura V.3).

DISCUSIÓN

160

Figura V.3. Alineamiento de PgChP con otras quitosanasas fúngicas y bacterianas. Los

aminoácidos sombreados son comunes a todas las quitosanasas del alineamiento. Las tres

zonas altamente conservadas en quitosanasas fúngicas están englobadas en cuadros.

*: Residuos de ácido aspártico y glutámico esenciales para la función catalítica.

Números de adquisición de las proteínas: XP_001390407 (A. niger), BAD08218 (A. oryzae-

1), XP_001824257 (A. oryzae-2), XP_754126 (A. fumigatus), NP_823026 (S. avermitilis) y

YP_001822750 (S. griseus).

PgChP --MMTYSRLIPFALFALFGTGLAQTVDGSKFNKPDGGPPGSYFAAGSSIPVAALQSAAAK A.niger MAFKTTAGLA---FLALAGSVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAAK A.oryzae-1 MPIKSFASRLALS-LAICGTAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK A.oryzae-2 MPIKSFASRLALS-LAICGTAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK A.fumigatus MPSTTIIRQLAIS-LALCNSALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK S.avermitilis MRTRTLTLAAAAGAALLATAALPATASPSGTKAPASA------QEGS-VSAASLLAKVTS S.griseus MRTRVLAL----TSAACCAALLGAAALPASATGPGAGQEPLAAEAGAPATAAELLAEVRA PgChP ARTPVPDATYPINGDNGAKKVTIHSDWAKFDKGAAYVWIADMDVDCDGIDYK-CKGNPD- A.niger ASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYK-CKGNGD- A.oryzae-1 ASKVPSHATYPIG--QGSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHG-CKGNPD- A.oryzae-2 ASKVPSHATYPIG--QGSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHG-CKGNPD- A.fumigatus ATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSG-CQGNPD- S.avermitilis CSQI-SNGKY--KTDDETS-ATI----PVCGKNGAVFWKADMDIDCDGQVTGKCNGTTDP S.griseus CSRI-SKGAY--RTDSGASRATV----PVCGTGDAVFWKADMDIDCDGRETKACSRKTDP PgChP -GQHQTNFGA-----LAAYEVPFFVIPD--RFGTKYAKQLPGNNVGAVIC----NGKMFY A.niger -GLPETNWGA-----LSAYEVPWIVIPD--QFLTANEDLLPGNNVAAVIC----NGKMYY A.oryzae-1 -GQKETNWGA-----LSAYEVPFIVIPQ--EFLDANKGTLKGNAVAAVIC----NGKMFY A.oryzae-2 -GQKETNWGA-----LSAYEVPFIVIPQ--EFLDANKGTLKGNAVAAVICATSSNGKMFY A.fumigatus -GQPQTNWGA-----LSAYEVPFIVIPD--KYLSANSGALPGNNIAAVIC----NGKMFY S.avermitilis WFQDDTAFHQSDGKPLRADSLPYVVVPSSSSIWNYASAGIKGGGVVAVIY----NNKVEY S.griseus YFLPETAFPSSRGEPLDSAVLPHVVVPGASKVWDYRRSGLTGGSVVAVVY----RDRVRY PgChP GIYGDSDGDTPQVIGEASWLMARTCFPNDDLNGNSGHGDVDVTYILFTGDDSVLPSSALN A.niger GILGDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALN A.oryzae-1 GIFGDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALN A.oryzae-2 GIFGDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALN A.fumigatus GILGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALT S.avermitilis AVVGDTG--PTKIIGEASYATAQALGIDPD--PETGGTDSGVTYILFKNS-QVSP----- S.griseus GVIGDTG--PTGIIGEASYAMAKTLGIDPD--PSTGGAESGVTYIAFKDS-RVSP----- PgChP KNYVTNFTTLRSMGDKLMTALAKNLKLVDGGDGGSP--TTTAGSNPT--SGSCEWEGHCA A.niger KNYITNFSTLRSMGDKLVGALASNLGLTSSA---------------SGSTATCSWQGHCE A.oryzae-1 KNYITNFDTLRSMGDSLVGALAKNLNLGGGGGNPPTTLTTTSIPEPTGGSGSCSWPGHCA A.oryzae-2 KNYITNFDTLRSMGDSLVGALAKNLNLGGGGGNPPTTLTTTSIPEPTGGSGSCSWPGHCA A.fumigatus KNYITNFTTLRSMGDKLVNALVSNLGLSGTPPTKTTTRVTTTTTKPT-SAASCSWAGHCL S.avermitilis ---IESHSAAVSLGDSLAKQFLQNN----------------------------------- S.griseus ---IESRARARSRGAERAREFVGR------------------------------------ PgChP -----GASCKDE------------------------------------------------ A.niger -----GAICSTEDDCSDDLVCDSGKCSSPDEDDGDDEDDEDEEENDEDDEDDDDE----- A.oryzae-1 -----GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCSDELACIS A.oryzae-2 GFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCSDELACIS A.fumigatus -----GASCSSDDDCADALVCTAGKCSVDGA-ATCSWEGHCEGASCSSD----------- S.avermitilis ------------------------------------------------------------ S.griseus ------------------------------------------------------------ PgChP --------------------------NDCSDQLVCKSGKCPSD A.niger --------------------------DDEDDE----------- A.oryzae-1 GICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA- A.oryzae-2 GICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA- A.fumigatus --------------------------DDCSDDLYCKSGSCTAP S.avermitilis ------------------------------------------- S.griseus -------------------------------------------

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DISCUSIÓN

161

De las quitosanasas fúngicas conocidas, sólo dos están producidas por mohos del

género Penicillium, concretamente P. islandicum y P. spinulosum (Fenton y Eveleigh,

1981; Ak y col., 1998), no habiéndose descrito hasta el momento ninguna quitosanasa

producida por P. chrysogenum. Por otra parte, aunque son frecuentes las glicosilaciones en

proteínas con actividad antifúngica purificadas a partir de plantas (Islam y col., 2002;

Deepak y col., 2003; Yamunarani y col., 2005), hasta el momento sólo se ha descrito este

tipo de modificación postraduccional en las quitosanasas de Mycobacter AL-1 (Hedges y

Wolfe, 1974) y P. islandicum (Fenton y Eveleigh, 1981).

Adicionalmente, tanto el peso molecular en torno a 30 kDa como el pI de 5

determinados para PgChP (Manuscrito 4), se encuentran dentro de los rangos descritos

para otras quitosanasas, de entre 10 y 50 kDa y pIs entre 4 y 10 (Somashekar y Joseph,

1996). La quitosanasa glicosilada con 30 kDa y pI 4,2 de P. islandicum (Fenton y

Eveleigh, 1981) es la más similar a PgChP desde el punto de vista bioquímico. No

obstante, hasta el momento se desconoce su secuencia aminoacídica, por lo que no es

posible establecer una homología entre ambas proteínas.

Por todo ello se puede afirmar que PgChP es una nueva quitosanasa de 30 kDa

producida por P. chrysogenum incluida en la familia GH-75. Además, esta proteína ha

mostrado actividad antifúngica frente a cepas de interés en alimentos (Acosta y col.,

2009a), lo cual no ha sido comprobado en otras quitosanasas.

En relación con PgAFP, el análisis de las secuencias tanto aminoacídica como

genética y su posterior comparación en bases de datos, permitió descartar la similitud con

PgChP. Así, PgAFP mostró homología con un conjunto de proteínas con actividad

antifúngica secretadas por mohos, concretamente: PAF de P. chrysogenum (Marx y col.,

1995), AFP de A. giganteus (Nakaya y col., 1990; Lacadena y col., 1995), Anafp de A.

niger (Lee y col., 1999) y AcAFP de A. clavatus (Skouri-Gargouri y Gargouri, 2008).

Estas proteínas tienen una estructura primaria diferente a la descrita en otras proteínas

antifúngicas producidas por plantas, invertebrados o anfibios, pero comparten

características comunes con ellas, como su pequeño tamaño (51-60 aminoácidos; 5,8-6,7

kDa), alto contenido en residuos de cisteína y un marcado carácter básico debido a la

presencia de lisina y arginina (Theis y Stahl, 2004). Adicionalmente, aunque hasta el

momento no se han descrito modificaciones post-traduccionales en este tipo de proteínas,

sí se han encontrado discrepancias en el peso molecular determinado mediante distintas

técnicas. Concretamente PAF corresponde a una banda de unos 12 kDa en SDS-PAGE

DISCUSIÓN

162

(Marx y col., 1995), pero a partir de la secuencia aminoacídica deducida del gen su peso

sólo se estima en 6,3 kDa (Marx, 2004); esta diferencia de masa también se aprecia en

Anafp y AcAFP (Lee y col., 1999; Skouri-Gargouri y Gargouri, 2008). Estos resultados

son similares a los expuestos anteriormente para PgAFP, si bien estas diferencias podrían

atribuirse, como se indicó anteriormente, a su marcado carácter catiónico (Mondstadt y

Holldorf 1990).

Por lo tanto, PgAFP no sólo muestra una gran similitud en sus secuencias genética

y aminoacídica con este tipo de proteínas antifúngicas producidas por mohos, sino que

también comparte otra serie de características comunes, ya que se trata de una proteína de

58 aminoácidos, con una masa molecular de 6,5 kDa y pI de 9,2. Además, tal como se

recoge en la tabla V.2, esta proteína presenta otras características comunes, como un alto

contenido de aminoácidos básicos y seis residuos de cisteína.

Tabla V.2. Parámetros de las formas maduras de las proteínas PgAFP, Anafp, PAF, AFP, y las

deducidas de Penicillium nalgiovense (NAF), A. niger, Neosartorya fischeri y Giberella zeae.

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Los seis residuos de cisteína presentes en PgAFP están conservados en todas las

proteínas antifúngicas de mohos (Marx, 2004), aunque AFP que posee dos residuos

adicionales (Wnendt y col., 1994). De hecho, la gran estabilidad mostrada por este tipo de

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DISCUSIÓN

163

proteínas frente a proteasas, altas temperaturas o pHs extremos (Lacadena y col., 1995) se

ha atribuido a la presencia de estos residuos, que estabilizan la estructura terciaria mediante

la formación de puentes disulfuro (Campos-Olivas y col., 1995).

Con respecto a las proteínas antifúngicas producidas por mohos, sólo han sido

caracterizados los genes que codifican AFP y PAF (Wnendt y col., 1994; Marx y col.,

1995). Asimismo, se ha descrito un gen homólogo al que codifica PAF en P. nalgiovense,

denominándose la proteína deducida NAF, aunque ésta no se ha purificado (Geisen, 2000).

El análisis de las secuencias de aminoácidos revela homologías del 31 al 42% entre PAF,

AFP y Anafp, mientras que PAF es 100 % idéntica a NAF. De las proteínas descritas y

purificadas, Anafp mostró la mayor similitud con PgAFP, concretamente un 79%.

Tal como ya se ha indicado en el apartado anterior para PgAFP, tanto AFP como

PAF son sintetizadas como preproproteínas, es decir contienen una secuencia péptido señal

para su secreción y una prosecuencia que es eliminada antes o durante su liberación al

medio (Marx, 2004). Con respecto a Anafp, aunque sólo se ha determinado la secuencia

aminoacídica de la proteína madura (Lee y col., 1999), se asume por su gran similitud con

el resto que también está codificada como preproproteína (Marx, 2004). En relación a la

proteína AcAFP sólo se han descrito los 21 primeros aminoácidos, de los cuales 20 y 12

residuos coinciden con la región amino terminal de AFP y PAF respectivamente.

En los últimos años, gracias a la secuenciación del genoma de diferentes mohos, se

han ido recogiendo en bases de datos proteínas deducidas que muestran similitud con este

grupo de proteínas antifúngicas. De hecho PgAFP, al igual que PAF, AFP y Anafp, ha

mostrado homologías con proteínas deducidas de N. fischeri y G. zeae (Manuscrito 2,

figura 6). Una de estas proteínas deducidas, concretamente del genoma de A. niger, ha

mostrado junto a Anafp una alta similitud con PgAFP, con un 75 y 79 % de homología,

respectivamente. Por otra parte, a pesar de que tanto PgAFP como PAF son producidas por

cepas de P. chrysogenum sólo mostraron un 43 % de homología, por lo que se trata de

proteínas diferentes. Por último, las secuencias que codifican las proteínas PgAFP y PgChP

se han localizado en el genoma de P. chrysogenum Wisconsin 54-1255 cuya secuencia

completa ha sido determinada recientemente (Van den Berg et al., 2008).

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164

V. 4. MECANISMO DE ACCIÓN DE LA ACTIVIDAD ANTIFÚNGICA DE PgAFP Y

PgChP

Conocer el mecanismo de acción de proteínas antifúngicas como PgChP y PgAFP

puede ser de gran utilidad para el desarrollo de nuevas estrategias antimicrobianas y para

determinar su posible aplicación en determinados productos.

Los mecanismos de acción de estas proteínas son tan variados como los organismos

que las producen, e incluyen degradación de polímeros de las paredes celulares fúngicas,

formación de canales o poros en la membrana, inactivación de ribosomas, alteración de la

síntesis de ADN o inhibición del ciclo celular (Selitrennikoff, 2001). No obstante, aún se

desconoce el modelo de acción de muchos de estos compuestos.

En cualquier caso, las proteínas antifúngicas de mayor utilidad son las que

presentan mecanismos de acción específicos para las membranas de los hongos (Groll y

col., 1998), ya que suelen ser poco tóxicas para las células de mamíferos. Los factores que

desempeñan un papel más destacado en la interacción del péptido con la membrana son la

carga (Hoover y col., 2003) y la hidrofobicidad del compuesto (Friedrich y col., 1999;

Hancock y Chapple, 1999). De hecho, el carácter catiónico se ha relacionado de manera

directa con la actividad antifúngica (Broekaert y col., 1992; Nielsen y col., 1997b; Koo y

col., 1998; Hong y col., 2001). La base de la especificidad de este tipo de proteínas

catiónicas es la composición lipídica de la membrana, ya que al estar cargadas

positivamente pueden interaccionar con los fosfolípidos aniónicos (Sitaram y Nagaraj,

1999) presentes en las membranas de bacterias y hongos. Por el contrario, en el caso de los

eucariotas superiores, esta unión es muy débil, ya que sus membranas están compuestas

principalmente por fosfolípidos sin cargas negativas (Theis y Stahl, 2004). El hecho de que

tanto PgChP como PgAFP muestren actividad antifúngica pero no produzcan hemólisis de

glóbulos rojos de mamíferos, puede apuntar a un mecanismo de acción específico para las

membranas de mohos.

Aunque la composición de la pared celular fúngica varía en función del género y la

especie, los polímeros más frecuentes son quitina, �1,3-glucano y �1,6-glucano. Otros

polímeros como el quitosán sólo se han descrito en un limitado número de hongos de los

géneros Rhizopus, Absidia, Fusarium o Mucor (Price y Storck, 1975; Alfonso y col., 1995;

Gao y col., 1995). Se ha atribuido un papel de defensa a las quitosanasas por su capacidad

de hidrolizar quitosán desestabilizando la pared celular (Somashekar y Joseph, 1996), y en

algunos casos se ha relacionado directamente la actividad antifúngica con la actividad

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165

quitosanasa (Saito y col., 2009). Sin embargo, distintos compuestos y tratamientos

térmicos provocaron efectos diferentes sobre la actividad quitosanasa y antifúngica de la

proteína PgChP, lo que podría significar que la actividad antifúngica no se debe a la

degradación del quitosán. Además, PgChP mostró actividad frente a P. commune, especie

en la que hasta el momento no se ha descrito la presencia de quitosán.

Por otra parte, tanto PgChP como PgAFP poseen carga neta positiva al pH de 4,5

empleado para evaluar la actividad antifúngica. Esta característica, junto a la capacidad de

formar estructuras anfipáticas, es común a todas las proteínas que actúan sobre las

membranas microbianas, aun presentando éstas estructuras primarias muy diversas y

secundarias tanto en hoja-� como en α-hélice (Tossi y col., 2000). Se ha descrito un

mecanismo de acción para algunas de estas proteínas, el modelo Shai-Mat-suzaki-Huang

(SMH), que propone la interacción de éstas con los fosfolípidos de carga neta negativa

presentes en la membrana plasmática de bacterias y hongos provocando la formación de

poros en la misma (Matsuaki, 1999; Shai, 1999; Yang y col., 2000). Entre estas proteínas

se encuentran algunas defensinas, que son un grupo de proteínas y péptidos

antimicrobianos producidos por mamíferos (Ganz y col., 1990), insectos (Lamberty y col.,

1999) o plantas (Broekaert y col., 1995), muy similares a las proteínas antifúngicas

producidas por mohos en cuanto a su pequeño tamaño, carácter catiónico y presencia de

puentes disulfuro en su estructura. Sin embargo las defensinas producidas por mamíferos e

insectos, de menor tamaño (3 a 5 kDa), forman canales iónicos en la membrana plasmática

provocando la pérdida de minerales y metabolitos esenciales (Lehrer y col., 1989; White y

col., 1995). Adicionalmente, algunas defensinas de plantas producen una desporalización

de la membrana como consecuencia de su unión a los fosfolípidos aniónicos (Thevissen y

col., 1999). No obstante, PgAFP no presenta actividad frente a bacterias y levaduras

(Acosta y col., 2009b). Estos datos coinciden con lo descrito hasta ahora para las proteínas

antifúngicas más similares a PgAFP, como PAF (Kaiserer y col., 2003), AFP (Lacadena y

col., 1995; Theis y col., 2003) y AcAFP (Skouri-Gargouri y Gargouri, 2008). Anafp en

cambio, es la única proteína de este grupo activa frente a levaduras (Lee y col., 1999), si

bien hasta el momento se desconoce su mecanismo de acción. La mayoría de estas

proteínas además de no mostrar actividad frente a levaduras y bacterias, manifiestan

diferentes espectros de inhibición frente a mohos (Marx, 2004). Por ello, tal como se ha

descrito para algunas defensinas de plantas (Thevissen y col., 1997; Thomma y col., 2002),

se ha propuesto un mecanismo de acción basado en la existencia de receptores específicos

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166

para cada proteína en las membranas plasmáticas de los mohos sensibles (Marx y col.,

2007; Meyer, 2008). Esta idea se ve apoyada por el hecho de que la actividad de PAF y

AFP es fuertemente inhibida en presencia de Mg2+ y K+ (Theis y col., 2003) y Ca2+, Na+ y

K+ (Kaiserer y col., 2003), respectivamente, ya que estos cationes podrían saturar los

receptores existentes en las membranas de los mohos sensibles impidiendo la unión de las

proteínas antifúngicas y con ello su actuación (Theis y col., 2003).

Al igual que ha sido descrito para PAF y AFP, tanto PgChP como PgAFP han

mostrado diferente sensibilidad a los cationes presentes en el medio. Así, aunque altas

concentraciones de Mg2+ y Ca2+ inhiben la actividad antifúngica de ambas proteínas, los

cationes monovalentes Na+ y K+ únicamente afectan a la actividad de PgAFP (Manuscritos

3 y 6). Concretamente, al combinar una pequeña cantidad de proteína (3µg/ml) con una

alta concentración de iones (100 mM) se aprecia una pérdida de actividad, aunque sólo

frente a A. niger. Por lo tanto, el mecanismo de acción de PgChP y PgAFP podría basarse

en la existencia de receptores específicos en las membranas de los mohos sensibles,

ausentes en levaduras o bacterias. Estos receptores tendrían mayor afinidad por los

cationes divalentes y además diferirían en función del moho sensible, siendo por ejemplo

los receptores para PgAFP en A. niger más afines a Na+ y K+ que los presentes en P.

restrictum.

Por otra parte, aunque el mecanismo de acción de PAF y AFP podría estar mediado

por la presencia de receptores específicos en la membrana, ambas proteínas ejercen efectos

diferentes sobre los mohos sensibles. Concretamente, la unión de PAF con el receptor

específico incrementa la permeabilidad al K+, provocando una hiperpolarización de la

membrana que conlleva la entrada de la proteína mediante endocitosis. A partir de ese

momento se activan distintos mecanismos, como la producción de especies reactivas de

oxígeno o mitoptosis (formación de poros en las membranas mitocondriales), que

ocasionan la muerte celular programada del moho sensible (Marx y col., 2007). A pesar de

las similitudes con PAF, el efecto de AFP es diferente, ya que sólo los mohos resistentes

son capaces de introducirla por endocitosis posiblemente para una degradación con fines

nutricionales. La unión de AFP a los receptores específicos en los mohos sensibles,

provoca tanto alteraciones en la membrana como la inhibición de la enzima quitín sintasa

que interviene en la biosíntesis de la pared celular fúngica (Hagen y col., 2007; Meyer,

2008). La proteína AFP posee además en su extremo amino terminal la secuencia de unión

a la quitina CKYKAQ (Hagen y col., 2007), no descrita en PAF, Anafp, PgAFP, ni PgChP.

DISCUSIÓN

167

Por todo ello, aunque la información disponible sugiere que el mecanismo de

acción de PgAFP y PgChP está basado en la interacción específica con receptores

presentes en las membranas fúngicas, sería necesario confirmarlo, y en su caso estudiar

tanto los receptores implicados, como el efecto que las proteínas ejercen tras la unión con

los mismos.

V. 5. APLICACIONES DE LAS CEPAS DE P. chrysogenum Y LAS PROTEÍNAS PgAFP Y

PgChP

Como ya se ha descrito en esta memoria, las condiciones ecológicas que ofrecen los

productos cárnicos madurados favorecen el desarrollo de una población fúngica

superficial, que aunque puede contribuir a la maduración (Martín y col., 2004, 2006),

también puede originar defectos o alteraciones (Sanz, 1986; Spotti y col., 1988) e incluso

producir micotoxinas (Núñez y col., 1996, 2007; López-Díaz y col., 2001; Sabater-Vilar y

col., 2003). Para controlar la población fúngica podrían utilizarse cultivos iniciadores que

al implantarse en el producto fuesen capaces de inhibir por competición a los mohos no

deseados. No obstante, la evolución de las condiciones ecológicas a lo largo de la

maduración puede hacer que los cultivos terminen siendo desplazados por otras cepas

mejor adaptadas. En cambio para los aislamientos de P. chrysogenum RP42C y AS51D, la

producción de las proteínas PgAFP y PgChP con actividad antifúngica, puede suponer una

ventaja adicional para competir frente al resto de mohos. De hecho, tanto PgAFP como

PgChP se purificaron como resultado de la búsqueda de mohos productores de proteínas

antifúngicas para su utilización como cultivos protectores en productos cárnicos

madurados (Acosta, 2006). Además, ambas cepas han sido aisladas de jamón curado, por

lo que se trata de mohos adaptados a las condiciones de temperatura, actividad de agua y

pH que se dan a lo largo del proceso de maduración. Adicionalmente, P. chrysogenum ha

sido propuesto como cultivo iniciador para la elaboración de productos madurados por su

contribución a la formación de sabor y aroma en el producto acabado (Martín y col., 2004,

2006; Benito y col., 2005). Dado que P. chrysogenum RP42C produce proteína activa en

condiciones similares a las de las etapas iniciales de la maduración (Acosta, 2006), será de

gran interés confirmar la producción tanto de PgAFP como de PgChP en productos

madurados. Además, el amplio espectro de inhibición mostrado fundamentalmente por P.

chrysogenum RP42C frente a mohos toxigénicos de interés en estos productos, como A.

flavus, A. parasiticus o P. commune, permite ser optimistas respecto a su utilización como

cultivo protector para garantizar la seguridad alimentaria en este tipo de alimentos.

DISCUSIÓN

168

No obstante, también podrían emplearse ambas proteínas purificadas en productos

en los que no sea deseable el desarrollo de mohos. Además PgChP, debido a su actividad

quitosanasa, podría también emplearse para la producción de oligómeros de quitosán a

nivel industrial.

De forma general y como paso previo a la aplicación de una proteína a nivel

industrial, resulta de gran importancia garantizar que la actividad antifúngica se mantiene

en presencia de determinados compuestos que puedan estar presentes en el producto donde

se vaya a aplicar (Theis y Stahl, 2004). Adicionalmente el efecto de ciertos tratamientos a

temperaturas o pHs extremos puede condicionar también sus posibles aplicaciones. Por

otra parte, la posibilidad de incrementar la actividad de estas proteínas mediante la adición

de determinados sustratos es otro factor a tener en cuenta antes de proponer futuras

aplicaciones.

V. 5. 1. APLICACIÓN DE PgChP Y PgAFP COMO AGENTES ANTIFÚNGICOS

Como se ha comentado anteriormente, no sólo resulta de gran interés la posibilidad

de utilizar mohos productores de proteínas antifúngicas como cultivos protectores en

productos madurados, sino que también puede ser útil el uso de PgAFP y PgChP como

aditivo en alimentos en los que no sea deseable el desarrollo del moho productor.

El uso de proteínas antimicrobianas como protectores en alimentos no es un

concepto nuevo. Concretamente la nisina, bacteriocina producida por varias cepas de

Lactococcus lactis, ha sido aprobada como GRAS (“Generally Regarded as Safe”) en ocho

países de la Unión Europea. Esta proteína es ampliamente utilizada en productos lácteos,

vegetales, carne o pescado (Delves-Broughton y col., 1996) para evitar el desarrollo de

bacterias patógenas gram positivas como Bacillus cereus, Listeria monocytogenes o

determinadas especies de Enterococcus, Streptococcus y Staphylococcus (Lubelski y col.,

2008). Además, la nisina ha sido comercializada para el tratamiento de infecciones

gástricas causadas por Helicobacter spp. y algunas de sus variantes están siendo probadas

en ensayos clínicos para tratar enfermedades producidas por enterococos resistentes a la

vancomicina (Reddy y col., 2004).

No obstante, antes de proponer a PgAFP y PgChP para su utilización como

compuestos antifúngicos en alimentos se deben evaluar los posibles efectos indeseables

que pudieran ocasionar en los consumidores para evitar cualquier peligro de tipo sanitario.

Por otra parte, la estabilidad de las mismas tras el tratamiento a diferentes temperaturas y

DISCUSIÓN

169

pHs o en presencia de determinados compuestos que se encuentren en los sustratos donde

se pretendan utilizar, condicionará en gran medida sus posibles aplicaciones.

Como se ha mencionado, tanto PgChP como PgAFP fueron inicialmente

purificadas mediante cromatografía de intercambio catiónico (Acosta y col, 2009a) con el

objeto de obtener proteínas con carácter básico, ya que esta característica está relacionada

con una mayor actividad antifúngica y una menor toxicidad para las células de mamíferos

(Hong y col., 2001). No obstante, si bien PgAFP mostró un pI de 9,2 (Acosta y col.,

2009b), PgChP mostró un pI de 5 (Manuscrito 4, figura 6), lo que supone que esta proteína

no es catiónica a pH fisiológico.

La ausencia de actividad hemolítica de PgChP y PgAFP frente a glóbulos rojos

(Manuscritos 3 y 6), y en el caso de PgAFP también de toxicidad para larvas de Artemia

salina (Manuscrito 3), permite ser optimistas hasta el momento con respecto a la inocuidad

de estas proteínas, ya que estas técnicas son comúnmente empleadas para chequear la

toxicidad de nuevas sustancias (Van’t Hof y col., 2001; Theis y Stahl, 2004; Milhem y col.,

2008; Chohan, 2009).

La ausencia de efectos negativos podría permitir la utilización de PgAFP y PgChP

en diversos campos de aplicación, si bien el presente estudio se ha enfocado hacia una

posible aplicación en alimentos. Por ello, se evaluó el efecto sobre la actividad antifúngica

tanto de diferentes tratamientos como de diversos compuestos comúnmente encontrados en

alimentos, como sales, detergentes o fosfatos (Tabla V.3; Manuscritos 3 y 6).

DISCUSIÓN

170

Tabla V.3. Resistencia de PgAFP y PgChP a tratamientos térmicos, distintos pHs, cationes, fosfato

sódico, quelantes y detergentes en las proteínas, expresada como los valores máximos a los que se

mantiene actividad antifúngica.

� � ��

� D� � �E:/�� $�� = �E:/� �� $��

�F � �G�� '��

HI� � ��

7 I� � ��

��I� � G� ��

:I� � �� G� ��

>� � � � /�� /��

>� � � � ��

�� 7 F �� G� �� G� � ��

� J �� K � GGG�

� J ��= � ��K � GGG�

� "$!��@ G� � GGG'� GGG�

L � L � ��&�� GGG�

� ��

M &��'��5$&14$&�� GGG� GGG�

a: Máxima concentración empleada 100 mM. b: Máxima concentración empleada 1 mM. c: Máxima concentración empleada 1 %. d---: Pérdida de la actividad antifúngica de la proteína. n.c.: Afectó al crecimiento del moho y su influencia no pudo determinarse.

n.d.: No determinado.

Muchos productos alimentarios se someten de forma rutinaria a tratamientos

térmicos, ya sea como consecuencia del propio proceso de elaboración o con el objeto de

garantizar su estabilidad microbiológica, lo que condiciona en gran medida la aplicación de

ambas proteínas. En este sentido, tanto PgChP como PgAFP mostraron actividad

antifúngica incluso tras tratamientos térmicos a 80 y 100ºC, respectivamente.

Dado que concentraciones de Ca2+ de 10 a 100 mM pueden inhibir la actividad de

estas proteínas, su uso se vería limitado en alimentos ricos en calcio como el queso. Si

embargo, la actividad de PgAFP y PgChP se mantuvo en presencia de altas

concentraciones de Na+ y K+, lo que puede ser de gran interés en productos cárnicos

madurados, donde abunda el cloruro sódico y puede haber otras sales de sodio y potasio.

DISCUSIÓN

171

Además, estas proteínas muestran una mayor resistencia a estos iones que otras como PAF

o AFP, cuya actividad ya se reduce en presencia de 40-60 mM de Na+ (Kaiserer y col.,

2003) o Na+ y K+ (Theis y col., 2003), respectivamente.

Otras sustancias como el EDTA y los fosfatos se utilizan como aditivos, entre otras

razones, por su capacidad de secuestrar metales en alimentos no ácidos que requieren

aditivos sin sabor (Calvo, 1991). Si bien los quelantes no tuvieron un gran impacto en la

actividad antifúngica de PgChP y PgAFP, ésta se redujo en presencia de una alta

concentración (100 mM) de fosfato, aunque esta disminución, en el caso de PgAFP,

depende de la concentración de la proteína y del moho sensible utilizado.

El uso de detergentes no iónicos como emulsionantes también está extendido en la

industria alimentaria. De hecho, en España se emplean en confitería, repostería y para la

elaboración de galletas (Calvo, 1991), si bien de los detergentes evaluados en este estudio

sólo tween 20 y tween 80 están autorizados en alimentos (E-432 y E-433,

respectivamente). Los detergentes inhibieron la actividad antifúngica de PgChP, pero

PgAFP se mantuvo activa con un 1% de tween 20 o de tween 80. Dado que esta

concentración supera los niveles habituales en los alimentos de 0,1-0,3% (R.D. 142/2002),

la presencia de estos aditivos no afectaría a la actividad de PgAFP.

Además de la utilización de PgAFP y PgChP de forma individual, la combinación

de ambas o con algún tratamiento físico o químico podría suponer un efecto sinérgico que

incrementara la actividad antifúngica en los alimentos. Adicionalmente, aunque PgAFP

haya mostrado un espectro de inhibición muy amplio (Acosta y col, 2009b), el uso

combinado con PgChP podría ampliar la actividad a mohos que posean quitosán en la

pared celular como Mucor o Rhizopus (Shimosaka y col., 2005; Somashekar y Joseph,

1996), géneros alterantes en productos cárnicos madurados.

Por otra parte, la estabilidad mostrada por ambas proteínas al tratamiento con

temperaturas y pHs extremos abre un gran abanico de posibilidades respecto de su

combinación con diferentes tratamientos físicos o químicos. La combinación de

compuestos antimicrobianos con tratamientos térmicos, químicos o físicos como las altas

presiones o los campos eléctricos pulsados, está siendo ampliamente estudiada. Así, la

nisina ha sido combinada con lisozima y campos eléctricos pulsados (Sobrino-López y

Martín-Belloso, 2008), con tratamientos de frío (Cao-Hoang y col., 2008), con calor y

acidificación (Naim y col., 2008), o con ácido láctico y monolaurina (Tokarsky y Marshall,

DISCUSIÓN

172

2008) para controlar diversos patógenos en alimentos. Combinaciones similares no sólo

pueden suponer un incremento en la actividad de PgAFP y PgChP, sino que también

podrían modificar su especificidad. De hecho la proteína antifúngica cecropina B, que por

sí sola no tiene efecto frente a E. coli, en combinación con lisozima es eficaz para combatir

este microorganismo (Casteels y col., 1993).

Por todo lo anterior, PgChP y PgAFP parecen tener características idóneas para ser

empleadas como agentes protectores en diversos alimentos que requieran un control frente

a mohos alterantes o toxigénicos. No es previsible que la utilización de estas proteínas

como aditivo provoque alteraciones en la flora intestinal beneficiosa del consumidor ya

que ambas poseen un mecanismo de acción específico frente a mohos. Por otra parte,

estudios realizados con PgAFP muestran la sensibilidad de esta proteína a tripsina, pepsina

y lisozima (Acosta, 2006), por lo que podría ser degradada en el tracto gastrointestinal.

No obstante, la aplicación de PgAFP y PgChP no tiene por qué estar limitada a un

uso en alimentos. De hecho se ha propuesto la utilización de proteínas antifúngicas

similares en otros campos como la agricultura o en terapéutica humana (Marx y col., 2007;

Meyer, 2008). En este sentido, la incorporación de genes que codifican proteínas

antifúngicas ha permitido la obtención de cultivos transgénicos resistentes a plagas de

hongos (Campo y col., 2008; Swathi-Anyradha y col., 2008) y ciertas proteínas

antifúngicas están siendo ya estudiadas para su uso como agentes farmacológicos

(Cappelletty y Eiselstein-McHitrick, 2007; Wiederhold y Lewis, 2007; Fortún y col.,

2009).

V. 5. 2. APLICACIÓN DE PgChP COMO QUITOSANASA

Dado que la caracterización aminoacídica de PgChP reveló que pertenecía a la

familia G-75 de quitosanasas, fue necesario comprobar su capacidad para hidrolizar

quitosán. Aunque algunas quitosanasas pueden hidrolizar polisacáridos distintos al

quitosán (Pelletier y Sygusch, 1990; Shimosaka y col., 1995; Kimoto y col., 2002), PgChP

no presentó indicios de degradación de la quitina (Manuscrito 6), como suele ser habitual

entre las quitosanasas (Fukamizo y Brzezinski, 1997; Su y col., 2006).

La quitina es el componente estructural mayoritario del exoesqueleto de

invertebrados y de las paredes celulares fúngicas (Lower, 1984; Ornum, 1992). Debido a

que la degradación de la quitina es muy lenta, la acumulación de desechos resultantes del

procesado de crustáceos es un problema emergente en la industria de productos de la pesca

DISCUSIÓN

173

(Shahidi y Synowiecki, 1991). No obstante, estos desechos pueden ser empleados para la

producción de sustancias muy beneficiosas a nivel industrial, como son la quitina, el

quitosán, o los productos de la degradación de los mismos. De hecho, los oligómeros

resultantes de la degradación del quitosán tienen múltiples aplicaciones: como inhibidores

de la producción de micotoxinas (Cuero y col., 1991), purificadores de agua (Jeuniaux,

1986; Muzzarelli y col., 1989), neutralizantes de acidez en zumos de fruta y vino (Chen y

Li, 1996; Rwan y Wu, 1996; Spagna y col., 1996), e incluso como agentes antitumorales y

potenciadores del sistema inmune (Pae y col., 2001). Adicionalmente, y como resultado de

la búsqueda de nuevos antimicrobianos naturales, los oligómeros del quitosán han

mostrado actividad antimicrobiana frente a bacterias, levaduras y mohos (Yalpani y col.,

1992).

El quitosán puede ser degradado mediante hidrólisis química o enzimática,

obteniéndose oligómeros de menor (monómeros-trímeros) o mayor grado de

polimerización (pentámeros-heptámeros), respectivamente. No obstante, los oligómeros

resultantes de una hidrólisis enzimática con quitosanasas muestran mejores características

funcionales (Kendra y Hadwiger, 1984; Uchida y col., 1988), lo que ha incrementado el

interés por este tipo de enzimas.

Dado que las quitosanasas difieren en sus propiedades enzimáticas (Wang y col.,

2008b), es necesario conocer el efecto sobre PgChP de diversos factores como iones o

detergentes para potenciar su actividad.

Los iones Ca2+ y K+ no tuvieron efecto alguno sobre la actividad quitosanasa de

PgChP a concentraciones de 100 mM, mientras que Mg2+ y Na+ sí influyeron en la

actividad por encima de 10 mM. En el caso de los quelantes, aunque la actividad se redujo

en presencia de una alta concentración (1 mM) de EDTA, con EGTA se llegó a triplicar la

cantidad de glucosamina liberada (Manuscrito 6). La influencia de altas concentraciones de

Mg2+ y Na+ sobre PgChP puede deberse a que los iones afecten a la estructura de la

proteína impidiendo su actividad, tal y como ha sido descrito para otras quitosanasas de

mohos (Wang y col., 2008b). De hecho, el tratamiento con SDS y ácido deoxicólico,

detergentes iónicos con conocido efecto desnaturalizante, produjo una inhibición de la

actividad de PgChP. Por el contrario los detergentes no iónicos, fundamentalmente el

Tritón X-100, favorecieron la actividad de la proteína. De forma similar, en presencia de

fosfato (10 mM) se duplicó la cantidad de glucosamina liberada.

DISCUSIÓN

174

En conclusión, la producción de oligómeros de quitosán mediante hidrólisis con

PgChP puede incrementarse en presencia de EGTA, Tritón X-100 y fosfato, siendo las

concentraciones más apropiadas 0,1 mM, 1 % y 10 mM, respectivamente. La proteína

podría además ser empleada en presencia de altos niveles Ca2+ y K+.

No obstante, la actividad quitosanasa de PgChP debe ser evaluada a diferentes

temperaturas y pHs con el fin de encontrar las condiciones óptimas para la degradación del

quitosán.

VI. CONCLUSIONES

CONCLUSIONES

175

VI. CONCLUSIONES

1. PgAFP es una nueva proteína antifúngica de 6,5 kDa, en la que no se han detectado

glicosilaciones. De acuerdo con su secuencia primaria, pertenece a un grupo de

proteínas antifúngicas de pequeño tamaño, básicas y con alto contenido en cisteína,

que son producidas por mohos.

2. La proteína antifúngica PgChP presenta diversas isoformas glicosiladas con pesos

moleculares comprendidos entre 30 y 31 kDa y, de acuerdo con su secuencia

aminoacídica y su actividad enzimática, es una nueva quitosanasa perteneciente a la

familia GH-75, mayoritariamente compuesta por quitosanasas fúngicas.

3. Las secuencias aminoacídicas deducidas de los genes que codifican estas dos proteínas

revelan la existencia de un péptido señal en ambas y una prosecuencia en PgAFP que

no están presentes en las proteínas purificadas.

4. La ausencia de actividad hemolítica y de toxicidad frente a larvas de Artemia salina,

junto con la reconocida inocuidad de P. chrysogenum, permite plantear la aplicación de

estas dos proteínas en alimentos.

5. Ambas proteínas antifúngicas muestran una gran estabilidad a tratamientos térmicos, y

mantienen su actividad en presencia de quelantes o fosfato sódico a las concentraciones

que pueden encontrarse en alimentos.

6. La actividad antifúngica de las dos proteínas sólo se ve limitada en presencia de altas

concentraciones de iones divalentes o de detergentes iónicos, si bien los detergentes no

iónicos afectan a la proteína PgChP. Por lo tanto, estas proteínas pueden ser de utilidad

en un amplio rango de alimentos.

7. La actividad quitosanasa de PgChP es independiente de la actividad antifúngica y no se

ve limitada por altas concentraciones de iones de calcio y detergentes no iónicos.

8. Las proteínas PgAFP y PgChP muestran características adecuadas para controlar el

crecimiento de mohos indeseables en alimentos.

CONCLUSIONES

176

VI. CONCLUSIONS

1. PgAFP is a novel antifungal protein of 6.5 kDa with no glycosylations. According to its

primary sequence, it belongs to a group of small, basic, and cystein-rich antifungal

proteins produced by moulds.

2. The antifungal protein PgChP has several glycosylated isoforms with molecular

weights ranging from 30 to 31 kDa. According to primary sequence and enzymatic

activity, it is a novel chitosanase belonging to the GH-75 family, mainly composed by

fungal chitosanases.

3. The putative amino acid sequences reveal a signal peptide in both proteins and one

prosequence in PgAFP that are not found in the purified proteins.

4. The lack of haemolytic activity and toxicity against Artemia salina larvaes together

with the safety granted to P. chrysogenum support the potential application of these

two proteins in foods.

5. Both antifungal proteins show a high thermal stability and keep their activity with

chelators or sodium phosphate at the concentrations found in foods.

6. The antifungal activity of the two proteins is only restricted by high concentrations of

divalent ions or ionic detergents, whereas non-ionic detergents only affect to PgChP.

Therefore, these two proteins can be of use in a wide range of foods.

7. The chitosanase activity of PgChP is not related to the antifungal activity, and it is

restricted by neither high calcium ion concentrations nor non-ionic detergents.

8. Both proteins show appropriate characteristics to control growth of unwanted moulds

in foods.

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BIBLIOGRAFÍA

177

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VIII. RESUMEN

RESUMEN

199

VIII. RESUMEN

Las condiciones ambientales en que se procesan o se almacenan diversos tipos de

alimentos favorecen el desarrollo en su superficie de mohos que tienen la capacidad de

adaptarse y desarrollarse en sustratos muy diversos. En productos cárnicos madurados y

quesos es frecuente el desarrollo una población fúngica superficial que puede tener efectos

deseables, ya que contribuyen en gran medida a la formación del sabor, aroma o textura del

producto acabado. Pero el desarrollo de mohos no siempre es deseable en alimentos.

Muchos mohos son alterantes, llevando al deterioro de las características sensoriales de los

alimentos. No obstante, el aspecto más preocupante respecto del desarrollo de una

población fúngica en alimentos es su capacidad para producir micotoxinas.

Existen numerosos mecanismos para controlar el desarrollo microbiano en distintos

tipos de alimentos, incluyendo métodos físicos y químicos. Estos tratamientos resultan

eficaces para evitar el desarrollo de mohos en determinados alimentos, pero no son

adecuados para otros productos como los madurados, donde la presencia de ciertos mohos

es beneficiosa y además la aplicación de fungicidas está limitada. Una posible solución en

este tipo de alimentos sería el empleo de proteínas antifúngicas que evitaran el desarrollo

de mohos indeseables y que a la vez permitieran el crecimiento de mohos beneficiosos en

los productos madurados.

En estudios previos se seleccionaron dos cepas no toxigénicas de P. chrysogenum,

RP42C y AS51D, procedentes de jamón curado, por su capacidad para inhibir el desarrollo

de mohos toxigénicos frecuentes en alimentos. Además, se purificaron dos proteínas con

actividad antifúngica, PgAFP y PgChP a partir de RP42C y AS51D, respectivamente.

El objetivo fundamental de este trabajo ha sido la caracterización de las dos

proteínas PgAFP y PgChP. Esta caracterización se ha basado en: la determinación de sus

secuencias aminoacídicas, la obtención de las secuencias de los genes que las codifican; la

evaluación preliminar de su toxicidad; el estudio de la influencia de diversos tratamientos

sobre la estabilidad de las proteínas y el efecto sobre la actividad antifúngica de

compuestos comúnmente encontrados en alimentos.

Pese a que tanto PgAFP como PgChP son proteínas con efecto antifúngico, han

resultado ser muy diferentes en cuanto a sus pesos moleculares, puntos isoeléctricos y

secuencias aminoacídicas. PgAFP tiene un peso molecular de 6,5 kDa, un punto

isoeléctrico determinado previamente de 9,2, y no posee glicosilaciones pese a que

RESUMEN

200

estudios preliminares apuntaban a que presentaba esta modificación. Este hecho se

confirmó con la obtención de la secuencia del gen que codifica la proteína y la

consiguiente determinación de la secuencia aminoacídica deducida. PgChP sin embargo,

mostró tener seis isoformas glicosiladas con pesos moleculares comprendidos entre 30 y 31

kDa, separados por una masa de 162 Da que podría corresponder a un residuo de manosa o

galactosa. Además, la secuencia deducida del gen permitió encontrar dos posibles sitios de

glicosilación (Asn-Phe-Thr y Asn-Asp-Cys) y la presencia de cadenas glucídicas se

demostró mediante deglicosilaciones enzimáticas. PgChP además presentó un punto

isoeléctrico cercano a 5, por lo que la proteína no es catiónica.

Las secuencias obtenidas de los genes pgafp y pgchp que codifican PgAFP y

PgChP tuvieron un tamaño de 276 y 912 pb, que codifican 92 y 304 aminoácidos,

respectivamente. Además, estos genes presentaron varios intrones pequeños con

marcadores típicos de intrones fúngicos.

La obtención de las secuencias aminoacídicas permitió encontrar proteínas con

cierta similitud en su estructura primaria. Así, PgAFP presentó un 79% de homología con

la proteína Anafp de A. niger y PgChP resultó tener cerca del 60% de similitud con la

familia de hidrolasas GH-75 compuesta por quitosanasas principalmente fúngicas. Tanto

PgAFP como PgChP presentan en su secuencia aminoacídica deducida una parte que

corresponde a un péptido señal necesario para que ambas proteínas sean secretadas al

medio extracelular. Además PgAFP, al igual que Anafp y otras proteínas con estructura

primaria parecida, presenta una prosecuencia, que parece mantener la proteína inactiva

para no causar daño a su moho productor, hasta que este fragmento es eliminado durante su

secreción.

La actividad antifúngica de PgAFP y de PgChP se inhibe por altas concentraciones

(100 mM) de ciertos cationes, especialmente los divalentes Ca2+ y Mg2+. Al igual que lo

propuesto para otras proteínas como las defensinas, el mecanismo de acción de PgChP y

PgAFP podría basarse en la existencia de receptores específicos en las membranas de los

mohos sensibles y ausentes en los mohos resistentes. Los cationes podrían saturar estos

receptores específicos e impedir que las proteínas se unieran a ellos y ejercieran su acción

antifúngica.

PgChP muestra actividad hidrolítica sobre quitosán, lo que junto a la homología de

su secuencia aminoacídica con otras quitosanasas fúngicas confirma que pertenece a este

RESUMEN

201

grupo de enzimas. Esta capacidad de PgChP para degradar quitosán puede hacer que su

espectro de inhibición incluya a hongos con quitosán en su pared celular, como los géneros

Rhizopus y Mucor, alterantes habituales en alimentos madurados. Además, las

quitosanasas son utilizadas en la industria para la degradación de caparazones de moluscos,

así como para la obtención de polímeros de quitosán, de alto valor como protectores frente

a patógenos en alimentos, inhibidores de micotoxinas, purificadores de agua, etc.

Por otro lado, es necesario demostrar la inocuidad antes de proponer la aplicación

de cualquier nueva proteína en alimentos. Aunque será necesario ampliar estos estudios,

PgAFP y PgChP no mostraron evidencias de toxicidad sobre glóbulos rojos y larvas de

Artemia salina a concentraciones superiores a las que poseen actividad antifúngica.

De forma general y como paso previo a la aplicación de una proteína a nivel

industrial, resulta de gran importancia garantizar que la actividad antifúngica se mantiene

en presencia de determinados compuestos que puedan estar presentes en el producto donde

se vaya a aplicar. Adicionalmente el efecto de ciertos tratamientos a temperaturas o pHs

extremos puede condicionar también sus posibles aplicaciones.

Ambas proteínas PgAFP y PgChP mostraron ser estables a altas temperaturas,

permaneciendo activas tras ser sometidas a 100 ºC 15 minutos y 80 ºC 20 minutos,

respectivamente. PgAFP además mostró ser estable a valores de pH entre 1 y 12.

La actividad antifúngica de PgAFP y PgChP se mantuvo en presencia de altas

concentraciones (100 mM) de sodio y potasio, lo que puede ser de gran interés en

alimentos madurados, donde abunda el cloruro sódico y puede haber otras sales de sodio y

potasio. Los quelantes EDTA y EGTA no tuvieron un gran impacto en la actividad

antifúngica de PgChP y PgAFP, mientras que el fosfato sódico a alta concentración (100

mM) sí pudo inhibir esta actividad. Los detergentes también inhibieron la actividad

antifúngica de PgChP, pero PgAFP se mantuvo activa a concentraciones de tween 20 o de

tween 80 más altas a las empleadas en los alimentos.

En definitiva, PgAFP y PgChP han mostrado alta estabilidad, han resultado ser

inocuas en los ensayos realizados y resisten componentes comúnmente encontrados en

alimentos. Aunque es necesario profundizar en su estudio, estas proteínas y las cepas de P.

chrysogenum que las producen muestran características adecuadas para su utilización

frente a mohos alterantes y toxigénicos en alimentos crudos madurados.

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