DR. ROBERTO PARRA SALDÍVAR

“Uso y producción de enzimas lacasas para aplicaciones en remediación de efluentes con contaminantes emergentes”

Magdalena Rostro-Alanisa, Leticia Ramírez-Cavazosa, Carlos Hernández Lunab, Roberto Parraa*a Centro de Biotecnología FEMSA, Escuela de Biotecnología y Alimentos, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico, 64849, Monterrey, NL, Méxicob Laboratorio de Enzimología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Av. Universidad s/n,Ciudad Universitaria San Nicolás de los Garza, NL 64450, México *r.parra@itesm.mx.

Emerging contaminants Chemicals in very low concentrations (ng / Lg / L) in wastewater, aquatic ecosystems and even drinking water supplies.

These compounds lack an established regulation and its health effects were unknown.

Recent studies have shown their carcinogenic, highly toxic implications in the etiology of some chronic diseases and high persistence in the environment.

Laccases (p-diphenol/ oxigen oxidoreductases, EC 1.10.3.2)

The mononuclear T1 site extracts electrons from the reducing substrate and mediates their

transfer to the trinuclear T2/T3 center where molecular oxygen is reduced (Bertrand, et.al., 2002)

The catalytic site is quite conserved, the rest of the enzyme structure shows high diversity

(Gochev, et. al., 2007).

Image S

ourc

e: S

olo

mon, E

. 2008

APPLICATIONS, CURRENT AND POTENTIALS

Waste Water

Treatment

Food Industry

Textile Industry (Dyes),

Pulp and Paper

(biobleachingand biopulping)

BiosensorsChemical Synthesis

The use of laccases in different fields has been studied in the last decades.

Laccases from Pycnoporus sanguineus strains

thermostability profiles, tolerance towards salts and organic solvents,

enzymes from a non-GMO could have food-grade status in many countries.

CS43 was able to degrade several dyes:

Remazol Brilliant Blue Reactive (anthraquinonic),

Acid Red 44 (azo),

Tripthenylmethane.

(Hernández-Luna, et al, 2008)

Their production levels reported are usually low or not as high as other no

thermostable wild laccases.

Selection of Strain

Hypothesis

P. sanguineus CS43 has the potential to be used in industrial scale laccase

production; and the produced laccases have interesting properties that have

advantages compared with other laccases previously reported in scientific literature.

Goals

1. Enhance the P. sanguineus CS43 laccase production and evaluate its scale up potential.

2. Characterize the laccases generated with the selected specific production conditions.

3. Perform a proof of concept over the degradation of the Endocrine Disrupting Chemicals (EDCs) nonylphenol and triclosan using the studied enzymes.

Screening

Design of Experiments

Duplicate runs in 10 L ST

Methodology Flow diagram

µ

Materials and Methods

Results

Fig. 1

Screening:

• TJM was selected for the highest laccase titers and low cost compared

with simmilar medium results (A).

• The co-inducer selected was SO with the highest activity measuerd (B).

From the results of the CCD, the ANOVA showed that the most significant factors in

laccase production (P≤0.001) were:

• The tomato juice medium concentration

• CuSO4 concentration

• the interaction of both concentrations

• Also, with a (P<0.05) the interaction of CuSO4 with soybean oil is important

Using a regression over the response surface, a prediction equation was generated

(Eq.1).

Y= 8514.1 + 15222.1*(TJM) - 6749.3*(CuSO4) + 5658.6*(TJM)^2 + 4246.4*(CuSO4)^2 -

3469.8*(TJM) (CuSO4) + 2080.2*(SO)(CuSO4)

Fig. 3 Plot of values observed versus predicted values of laccase activity at culture day 14 from the DOE experimental and model. (r2=94%, P<0.001).

A C

B

Fig. 4 Response surface models for laccase activity at culture day 14 under DOE experimental conditions. X and Y axis variables: (A) Tomato juice medium (TJM) and CuSO4, (B) SO and CuSO4

(SO) and (C) TJM and Soybean Oil.

Fig. 5 Confirmation of best treatment conditions for laccase activity. (A) Laccase levels observed from repetition of the best treatment condition of DOE (A00): () 36.82% (v/v) TJM, 1% (v/v) SO and CuSO4 3 mmol/L. () 36.82% (v/v) TJM no induced. (B) Enzyme activity profile at stirred tank bioreactor level. () Best stirred tank bioreactor run at A00 conditions. Highest activity registered about day 14 () typical pH profile read at secondary axis () flasks at A00 conditions.

The native strain CS43 of P. sanguineus is an ideal candidate for over-production of highly thermostable

laccases in an inexpensive medium composed of

Tomato juice

CuSO4

Soybean oil

The laccase production was successfully enhanced and scalable.

Increment in laccase activity up to 13 times compared with an optimized GP medium.

The cultivation time (at highest laccase activity) was reduced from 28 to 13 d, with a total

productivity increment of 26 times.

To the best of our knowledge, the levels of laccase activity achieved by P. sanguineus CS43 in

stirred tank bioreactor runs (143 000 IU/L) are the highest reported in the literature for this strain (Vanhulle et al., 2007; Dantán-González et al., 2008; Vite-Vallejo et al., 2009; Uzan et al., 2010; Lomascolo et al., 2002; Van Der Merwe, 2002;

Litthauer et al., 2007).

Conclusions

The model from the CCD indicated that laccase titers could be

significantly enhanced by increasing the concentration of the tomato

juice in the medium.

Also, a reduction of the levels of CuSO4 and/or Soybean oil needs to be

tested in future experiments as it is indicated by the model to achieve

higher laccase levels.

Conclusions

Products

Characterize the generated laccases.

Proof of concept degradation of EDCs

Mat &

Met: L

accase P

urific

atio

n

Culture supernatant from the 10 L STR enhanced production, collected at day 15

Filtration (200 micrometer mesh) + flow filtration (5-10 microns)

Freezing and Thawing

Flow filtrations (0.5 + 0.2 microns)

Ultrafiltration 10,000 MWCO

Anionic IEX (QFF HiTrap 5 ml GE). pH 5, 2 steps elution. Two peaks collected, one for each MW form of laccase.

HIC (Phenyl HP, 5ml GE) Gradient elution. One peak collected at around 0.5 M Na2SO4

Mat & Met: Photometric methods

Protein determinationbicinchoninic acid assay kit (QuantiPro BCA Assay Kit, Sigma, USA) using BSA as standard

UV-visible absorption spectrum each laccase recorded between 300 and 700 nm

Ma

t &

Me

t: C

ha

racte

riza

tio

nOptimal condition

pH:

from pH 2 to 8 (B&R buffer)

with 0.3 mM ABTS, 0.3 mM DMP and 0.3 mM guaiacol

@25°C

Temp:

from 25 to 75 °C. measured inmediatelyafter adding laccase

@ pH3 with ABTS .

Stability

pH:

Tested from pH 2 to 8.0 (B&R buffer)

@ room temperature.

Temp:

Tested from 40-70 °C.

@ pH7 Phosphate buffer,

Kinetic Parameters

Km and Kcat:

for ABTS (0.5 µM to 100 µM)

guaiacol ( 20 µM to 1000 µM)

DMP ( 20 µM to 1000 µM)

@ their optimal pH

calculated from NLSF regressions using Michaelis Menten model (Statistica, Softstat, USA)

All experiments were performed in three replicate. The initial laccase activity was around 200 U/L

Stability: Inactivation modeling

The pH (acidic region) and thermal inactivation through time was

modeled using the 3-parameter model of Aymard and Belarbi (2000)

(Ladero, Ferrero, Vian, Santos, & Garcia-Ochoa, 2005).

(1)

The ratio (A)t/(A)0 represents the enzyme activity remaining after time t.

α and β are complex expressions of rate constants.

C constant indicates the weight between both exponential parts of the equation.

The values were obtained by curve fitting of the plot of the residual enzyme activity versus

time using GraphPad Prism 6.0 software.

Half-life values were calculated accordingly to the equation (1) at (A)t/(A)0 =0.5.

Mat

and M

et:

Inh

ibitio

n a

nd D

egra

dation s

tudie

s

Inhibition Study

NaF, NaN3, urea, Na2SO4, NaCl, EDTA, acetonitrile (ACN), DMF, acetone, ethanol and Dimethyl sulfoxide (DMSO).

Incubated for 5 min

@ room temperature

Measured with ABTS @ pH3.

Degradation of EDCs: Nonylphenol (NP) and Triclosan (TS)

Samples prepared in distilled water with:

Culture Supernatant with 100 U/L of Laccase activity

10 ppm of each EDC

@ pH 5

HPLC 1200 system with XDE-C18 column.

Thermostated at 40 °C.

275 nm for NP and 229 for TCS

Flow 1 mL/min, injection volume of 20µL

Gradient of CAN and 10mM phosphate buffer pH5.

Inhibition Study experiments were performed in three replicates and the degradation profile was executed twice.

• For degradation comparison, an efficiency value was

calculated as the

concentration of analyte degraded, in parts per billion,

the laccase activity per Liter, the hours of treatment

Results

Several Isoforms presented

•Molecular mass estimated by SDS-Page:

85, 68 and 66 kDa

•The pI of 68 and 66 are around 4.5-4.7 by 2D

Gel.

Other

Laccase

Other

laccase

Lac I and

Lac II

Table 1. Purification of the P. sanguineus CS43

laccase isoformsPurification Step Volume

(ml)Total

protein (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Purification (fold)

Culture filtered 1178.6 1998.86 53591 26.8 100 1.0

Ultrafiltration 7.0 758.94 38212 50.3 71 1.9

IEX:

LacI 8.3 31.67 2284 72.1 4.3 2.7

LacII 9.1 250.96 14812 59.0 27.6 2.2

Total 17096 60.8 31.9 2.3

HIC:

LacI 1.6 6.37 1784 279.9 3.3 10.4

LacII 2.1 34.85 10137 290.9 18.9 10.9

Total 11921 289.3 22.2 10.8

Purification with IEX

QFF HiTrap 5ml, pH5.

Laccases were eluted with a two-step protocol at 3.5% and 10% of 1 M Na2SO4 at a flow rate of 5 mL min-1.

IEX was run at pH 5.0 with 0.2 M piperazine buffer.

0

20000

40000

60000

80000

100000

120000

0

500

1000

1500

2000

2500

3000

3500

4000

0 50 100 150 200 250 300 350 400

Ab

sorb

ance

at

28

0n

m

Volumes through the column (ml)

Typical Profile for IEX pH5 two steps

UV

Activity (U/L)

Act

ivit

y (U

/L)

Lac I

Lac II

Purification with Hydrophobic Interaction Column

Each fraction selected from IEX was process separately in a HIC Column. More active

fractions were selected for characterization studies.

Lac ILac II

Deglycosilation study

Fig. 1 Electrophoresis of laccase isoforms

from P. sanguineus CS43. Lane M Protein

standard; lanes 1 and 3 purified LacI and LacII

respectively; lanes 2 and 4 deglycosylated

isoforms LacI and LacII.

M 1 2 3 4

55

66

97

116

158

212

42

KDa

UV/Vis spectrum of purified P. sanguineus

laccases

Fig. 2 LacI (blue) and LacII (red)

isoforms. Measurements were

performed in triplicates.

The laccase exhibited an absorbance

peak at 610 nm , indicates the presence

of a type 1 copper atom in the molecule.

A shoulder at around 330 nm in this

spectrum suggests the presence of T3

copper site (Shleev et al., 2004).

Sequencing

Table 2. Amino acid sequence of five internal peptides from P. sanguineus CS43 LacI and LacII

isoforms. The underlined amino acids correspond to the differences between the two laccase

isoforms

Amino acid sequence

Peptide LacI LacII Identity (%)

1 FPNGADATLINGLGR FPPGSDATLINGLGR 87

2 TPGTTSADLAVIK SPGTTAADLAVIK 85

3 YSFVLDASQPVDNYWIR YSFVLDASQPVDNYWIR 100

4 ANPPFGNVGFAGGINSAILR ANPSFGNTGFAGGINSAILR 90

5 SAGSSEYNYDNPVFR SAGSSEYNYDNPIFR 93

Optimal conditions

Fig. 3 pH dependency on (A) LacI and (B) LacII

activities with ABTS (♦), DMP (▲), and guaiacol

(+) as substrates at 25°C. (C) Effect of

temperature on laccase activity with ABTS as

substrate at pH3; Lac I (♦), Lac II (■). Data

points represents the means of three replicates

± SD.

A B

C

pH

Sta

bility

Results

Fig. 4 Stabilities of LacI (A), LacII (B) and CS (C) at different pH; pH 2 (▼), pH 3 (♦), pH 4 (∆), pH 5(*), pH 6 (●), pH 7 (■), pH 8(▲). Curves between data points (solid lines) were fitted by least squares regression to a biexponential function. Dotted straight lines are connecting the dots without regression. Laccase activity was measured using ABTS as the substrate at pH 3. Data points represent the means of three replicates ± SD.

A B

C

Therm

osta

bility

Results

Fig. 5 The thermal stability at pH 7 of Lac I(A), LacII (B), CS (C); 40°C (▼), 50°C (♦), 60°C (■), 70°C (▲), 80°C(●). Laccase activity was measured using ABTS as the substrate at pH 3. Data points represent the means of three replicates ± SD.

Table 3. Half Life at specific temperature

or specific pH

Parameter: R2 Half-life (h)

CS LacI LacII CS LacI LacII

pH

2 0.9926 0.9931 0.9946 5.17 7.44 1.52

3 0.9971 0.9918 0.999 31.6 18.29 2.68

4 0.9876 0.9912 0.9979 81.75 24.89 2.49

5 0.9913 0.9943 0.9959 75.29 33.3 2.87

6 0.98 0.9724 0.9867 290.45 263.32 105.02

Temp C

40 NA NA 0.8984 >1012 >1012 1012

50a 0.9862 0.9733 0.9956 73.64 277.77 35.84

60 0.9772 0.9689 0.98 3.25 18 2.25

70 0.9996 0.9917 0.9987 0.18 0.64 0.14

80 0.9984 0.9991 1 0.02 0.03 0.01

Commercial available laccases as the ones produced by Trametes versicolor or Pleourotus ostreatus, do not share the thermostability capacity demonstrated by LacI.

Stoilova et al., (2010) reported a laccase from Trametes versicolor with an approximated half-life of 6 h at 50°C and 3 h at 60°C.

In Han et al., (2005) study, a half-life for same species laccase was less than 1 h at 60°C.

Rancaño et al., reported half-lives of 75 h and 5 h for 50°C and 60°C respectively (2003).

For Pleurotus ostreatus, Palmieri et al, reported for laccases POXA1w, POXA2 and POXC, half-lives of 3.3, 0.2 and 0.5 h respectively at 60°C (1997),

while Okamoto et al(2000). , and Karp (2012) reported half-lives of 20 and 8 minutes respectively at the same temperature

The thermostability of LacI at 60 °C (18 h) and 70 °C (0.64 h) compete in the high end range with others laccases from the genus Pycnoporus already reported, and to the best of our knowledge, the half-life of LacI at 50°C is from the highest reported for purified P.sanguineus laccases (277.7 h)

Discussion

Table 4. Michaelis-Menten kinetic constants of studied P. sanguineus laccases at their optimal pH.

Substrate Km (µM) Kcat (s-1)Kcat/ Km (mM−1 s-1

)Optimal pH

Lac I Lac II Lac I Lac II Lac I Lac II Lac I Lac II

ABTS 6.9 12.2 519.2 447.2 74816.4 36746.1 2.5 2

DMP 89.2 191.6 184.6 155.6 2068.4 812.2 3.5 3

Guaiacol 1484.5 1100.8 2472.5 2084.4 1665.5 1893.5 4 4

Table 5. The effect of inhibitors on purified laccase activity at 25°C. Laccase activity was measured using ABTS as the substrate at pH 3.

Inhibitors IC50 (mM or %a)Complete inhibition

(mM or %a)

LacI LacII LacI LacII

NaF 0.08 0.02 16 8

NaN3 6.2E-06 6.9E-07 16 16

Urea 5810 3930 >8000c >8000 c

Na2SO4 >800b >800b >800 c >800 c

NaCl 65 14 >2000 c >2000 c

Acetonitrilea >64b 33 >64 c >56 c

DMFa 37 26 64 64

Acetonea 47 31 >72 c >72 c

Ethanola 55 52 >64 c >64 c

DMSOa 41 33 72 72

EDTA 49 42 100 80

Degradation of EDCs

Fig. 6 Degradation profile in aqueous phase with 100 U/L of laccase activity from culture

supernatant, and 10 ppm of EDC at pH 5. (A) NP. (B) TCS. Data points represent the means

duplicates ± SD.

Co

nce

ntr

ació

n (

áre

a/ár

ea

inic

ial)

Co

nce

ntr

ació

n (

áre

a/ár

ea

inic

ial)

Tiempo (h)Tiempo (h)

A

Degradation of EDCs

The efficiencies obtained in the degradation of NP and TCS at pH5 and 25°C

(23.6 and 15.3 ppb*L/U*h respectively) were among the range for NP and high

for TCS in comparison with similar studies with other fungi laccases. (Cabana et

al., 2007; Kim and Nicell , 2006; Murugesan et al. 2010; Saito et al. 2004)

The use of the supernatant instead of the purified laccase can be the base to

develop sustainable methodologies with similar efficiency towards pollutant

degradation than with the purified enzymes while eliminating expensive

purification steps.

Conclusions

CS and the purified LacI and LacII isoforms obtained from the P. sanguineus CS43,

have unique properties that make them ideal for industrial applications as wastewater

treatment.

It was possible to effectively produce them with a cheap tomato juice base

medium on a 10 L bioreactor as stated in the previous work;

they have shown to be especially ideal for applications were high temperature

and/or organic solvents are present.

The have outstanding Michaelis-Menten kinetic parameters for typical substrates

They have high biodegradation capability proved for a pair of EDCs.

Products

Laccases Inmobilization

Advantages and Disadvantage of Immobilization

Hypothesis

Systems immobilized laccase P. sanguineus

that have better physicochemical properties

free enzyme.

Supports

Eupergit

Immobead

Lentikats

Immobilization testPreliminar test with commercial enzyme

According to the preliminary tests it was determined that in order of efficiency, it is considered bettersupport the Lentikats> Eupergit> Immobead.

In addition to assessing immobilization techniques for subsequent and final tests Covalent Link methodwas used because the results obtained show good results.

Immobilization test

Immobilization yields using 25 unitsof enzyme from Pycnoporussangineous applying the techniqueof covalent bonds for Eupergit andImmobead while for Lentikats wasfollowed with the manufacturer'sprotocol.

Enzimatic Activity at different pH

In this graph we can see that there is a shift in the performance of the enzyme when it is immobilized, achieving improve between the ranges of pH 3-5 for the case of Lentikats and Eupergit, while the immobead achieves its maximum activity at pH 3.

Enzimatic Activity at different temperature

In the graph it can be seen that in all cases greater activity was achieved at 70° C for the case of Eupergit and immobead, while due to the plastic nature of Lentikats presents deformation to 55° C so that it was impossible to test at higher temperatures.

Reusability test

After 5 cycles of use can see that there is wear on the catalytic capacity of the supports, according to the results obtained by the performance is as follows: Lentikats> Immobead> Eupergit

Reusability test

* Each cycle was performed saturating ABTS and 0.5 mM citrate phosphate bufferA pH in the optimum of each bracket 25 ° C

Conclusions

The laccases immobilized systems and Eupergit Pycnoporus sanguineus, and Lentikats Immobead supports possess better than free enzyme physicochemical and mechanical properties.

The best method of immobilization for Eupergit and Immobead supports is that of covalent bonds and the best method of immobilization for Lentikats is entrapment.

The enzyme-support systems tested have a satisfactory operation range pH between 2-6 and temperature from 25 ° C to 70 ° C, retaining a high percentage of enzyme activity.

Reusability tests showed the value of fixed assets, compared to only use they have free enzymes systems. Immobead Eupergit and retained enzyme activity 86% and 89% respectively at 5 cycles of use and Lentikats retained 89% of its enzymatic activity after 10 cycles.

Conclusions

Our research group is already working

in the characterization of the degradation of different water micropollutants,

in the complete sequencing of the isoforms to understand the differences that confers LacI a markedly better performance than LacIIin pH and thermal stability as well as better resistance to inhibitors.

Application Testing

Supports Economic functionalization

Scaling catalytic processes

1 Professor3 Postdocs

3 PhD candidates5 MSc candidates

5 Specialists10 Undergraduate students

Exchange students: Germany, Ecuador, Spain

30 people

Thank you!!!

Contact:

Dr. Roberto Parra SaldívarTecnológico de Monterrey, Campus MonterreyCEDES 405-ITelephone: +52 (81) 83582000 ext. 5561r.parra@itesm.mx