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8/13/2019 Producción Fitasa Por Aspergillus ficuum Y Otros En SSF

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Pro du ct ion of phytate hydrolys inge n z y m e b y s o m e fu n g i

S J H o w s o n a n d R P D a v i s

AR C Food R esearch Institute, Colney Lane, Norwich NR 4 7UA, UK

( R e c e i v e d 9 F e b r u a r y 1 9 8 3 )

Eighty-four fungi from twenty five species have been examined for the production of extracellular

enzymes capable of hydrolysing phytate 3-phytase, myo-inositol hexakisphosphate 3-phospho-

hydrolase, EC 3.1.3.8, and 6-phytase, myo-inositol hexakisphosphate 6-phosphohydrolase, EC 3.1.3.26)

when grown in: 1) rapeseed meal RSM); 2) a semisynthet ie medium containing phytate as the sole

phosphorus source PSM); 3) potato dextrose broth PDB). Although 58 active strains showed sub-

stantial activity, results in either of the media were of no value in indicating activ ity in RSM. There

was no relationship between the ability o f a fungus to hydrolyse phyt ate and its taxonomic position.

Asper g i l lus f i cuum NRRL 3135 had the greatest activity in the synthetic medium, and was relatively

active in RSM. The extracellular enz yme had max imum activity aft er 10 days growth in PSM and had

a temperature opt imum of 55°C. Two pH optima were noted at pH 2.0 and 5.5. Inorganic phosphate

inhibited enzyme production; ammonia ions were a better nitrogen source than nitrate or urea.

Keywords: Rapeseed meal; phytate; p hytate hydrolysing enzyme;Aspergillus ficuum

I n t r o d u c t i o n

S a l t s o f phy t ic ac id myo-inositol h e x a k i s d i h y d r o g e n -

p h o s p h a t e ) a r e f o u n d i n m a n y p l a n t t i s s u es a n d r e l a te d f o o d

p r o d u c t s . 1,2 P h y t a t e c o n s t i t u t e s a p p r o x i m a t e l y 1 - 2 b y

w e i g h t o f c e re a l s a n d o i l se e d s a a n d p h y t a t e p h o s p h o r u s

a c c o u n t s fo r b e t w e e n 6 0 a n d 9 0 o f t h e t o t a l p h o s p h o r u s

pr esen t in these s eeds ;4 i t is cons ide r ed to ac t e i the r a s ar e se r v e o f p h o s p h o r u s o r myo4nositol o r i n m a i n t a i n i n g

a p h o s p h o r u s b a l a n c e , s ' 6 T h e i n s o l u b l e p h y t a t e s a r e c l o s e ly

as soc ia ted wi th p r o te ins , e spec ia l ly in o i l s eeds7 and r educe

the b ioava i lab i l i ty4 ' 8 o f bas ic am ino ac id g r oups in the

pr o te ins . P hyt ic ac id i s a l so a s t r ong che la t ing agen t and

r e a d i ly b i n d s c o v a l e n t m e t a l i o n s , re n d e r i n g t h e m i n s o lu b l e

a n d t h u s u n a v a i la b l e f o r a b s o r p t i o n . 3 '9 T h e n u t r i t i o n a l c o n -

s e q u e n c e s o f p h y t i c a c i d i n t e ra c t i o n s w i t h p r o t e i n a n d

m i n e r a ls a r e w e l l d o c u m e n t e d 3 '4 ' 9 a n d m e t h o d s f o r i ts

r e m o v a l f r o m f o o d s o f p l a n t o r i g i n a r e d e s ir a b le . M o s t

r e s e a rc h h a s c o n c e n t r a t e d o n t h e r e m o v a l o f p h y t a t e f r o m

s o y a b e a n a n d r e l a t e d p r o d u c t s , b e c a u s e o f t h e i r i m p o r t a n c e

a s so u r c e s o f e d i b l e o i l a n d a s a p r o t e i n s o u r c e f o r h u m a na n d a n i m a l c o n s u m p t i o n . 3 A °

R e c e n t l y , i n c r e a s ed i n t e r e s t h a s b e e n g i v e n t o r a p e s e e d ,

t h e m o s t s u i t a b l e o i l s e e d f o r t e m p e r a t e c l i m a t e s a n d

c o n s e q u e n t l y a s o u r c e o f p r o t e i n ,n - 1 4 H o w e v e r , r a p e s e e d

m ea l and pr o te in i so la te s pos ses s even h ighe r phy t ic ac id

l e v el s t h a n m a n y p l a n t f o o d s a a n d a n i m a l f e e d i n g t r ia l s

h a v e h i g h li g h t e d t h e p r o b l e m s c a u s e d b y p h y t a t e , s - 1 7

M e t h o d s h a v e b e e n d e v e l o p e d f o r th e r e m o v a l o f p h y t a t e s

f r o m s o y a b e a n m e a l 4 a lt h o u g h n o n e h a s y e t a c h i e v e d

e c o n o m i c f e a s i b i l it y . O n e p o s s i b i li t y is t h e u s e o f p h y t a s e

e n z y m e s ( 3 - p h y t as e , rnyo-inositol h e x a k i s p h o s p h a t e

3 - p h o s p h o h y d r o l a s e , E C 3 . 1 . 3 .8 , a n d 6 - p h y t a s e , myo-inositol

h e x a k i s p h o s p h a t e 6 - p h o s p h o h y d r o l a s e , E C 3 . 1 . 3 . 2 6 ) f o r t h e

h y d r o l y s i s o f th e p h y t i c a c i d i n t o p h o s p h o r i c a c i d a n d

myo-inositol. S e v e ra l w o r k e r s h a v e s t u d i ed t h e p r o p e r t i e s

o f p h y t a s e e n z y m e s , i n c lu d i n g t h o s e i s o l a t e d f r o m m i c r o -

o r g a n i s m s . 1 8 - 2 ° I n d e e d , p h y t a t e r e d u c t i o n i s a c h a r a c t er i s ti c

o f f e r m e n t a t i o n s s u c h a s th o s e c a r r i ed o u t b y b a k e r s 'y e a s t 2 1 -2 3 a n d i n t h e p r o d u c t i o n o f s o y a b e a n t e m p e h . 24

T h e w o r k i n t h is l a b o r a t o r y w a s p r i m a r i l y c o n c e r n e d

w i t h t h e r e m o v a l o f p h y t a t e f r o m r a p e s e e d m e a l b y u s in g

p h y t a s e . T h i s p a p e r r e p o r t s a s u r v e y o f m i c r o o r g a n i s m s

f o r t h e a b i l i t y t o p r o d u c e a s u i ta b l e e x t r a c e ll u l a r p h y t a s e ,

s o m e c h a r a c t e r i s ti c p r o p e r t i e s o f a p a r t i a ll y p u r i f i e d m o u l d

p h y t a s e a n d a c o m p a r i so n o f t h i s e n z y m e w i t h w h e a t

6 - p h y t a s e .

M a t er ia l s a nd met ho ds

Organisms

C u l t u r es w e r e m a i n t a i n e d o n D i f c o - B a c t o y e a s t m a l t

a g a r ( Y M ) s l o p e s, e x c e p t f o r s t r a in s o f Botrytis cinerea

a n d Cladosporium s p p . , w h i c h w e r e k e p t o n D i f c o - B a c t op o t a t o d e x t r o s e a g a r ( P D A ) . T o i n c r e as e s p o r e p r o d u c t i o n ,

species of Aspergillus w e r e g r o w n o n C z a p e k - D o x a g a r.

Screen ing program me

T h e o p t i m u m m o i s t u r e c o n t e n t f o r t h e p r o d u c t i o n o f

p h y t a s e b y g r o w i n g c u l t u r e s i n r a p e s e e d m e a l w a s d e t e r -

m i n e d w i t h Rhizopus oligosporus, a m o u l d u s e d i np r o d u c i n g t e m p e h i n I n d o n e s i a 2 s a n d a l r e a d y s h o w n t o

r e d u c e t h e l e v e l o f p h y t a t e d u r i n g f e r m e n t a t i o n . 2 4

S p o r e s o f R . oligosporus w e r e h a r v e s t e d f r o m Y M

slopes , p r ev iou s ly incuba te d f o r 7 days at 25°C , us ing

9 m l 0 .1 T w e e n 8 0 . T h e s u s p e n s i o n w a s f i lt e r e d

t h r o u g h t w o l a y e r s o f s t e ri l e m u s l in , c e n t r i fu g e d a t

0141 --0229/83/050377--06 03.00© 1983 Butterworth & Co. (Publishers) Ltd Enzyme Microb. Technol. , 1983, Vol . 5, September 377



P a p e r s

Figure 1 P la tes show ing grow th o f a) Aspergillus niger b) A f i cu u m c) Saccharomyces cerevis iae and d) Geot r ichum

c n d i d u m on phyt se screening g r

4 0 0 0 r e v / m i n f o r 1 5 m i n , t h e s u p e r n a t a n t d e c a n t e d a n d

the spores re suspe nded in s t e r il e glas s d i s ti l led w a te r

( G D W ) . T h e s p o r e c o n c e n t r a t i o n w a s a d ju s t e d t o

2 × 1 0 6 m l - ~ .

D u p l i c a t e s a m p l e s ( 5 0 g ) o f r a p e s e e d m e a l w e r e

w e i g h e d i n t o 2 5 0 m l c o n i c a l f l as k s an d i n o c u l a t e d w i t h

1 0 m l o f s p o r e s u s p e n s i o n , gi vi ng a m o i s t u r e c o n t e n t o f

~ 2 5 . S t e ri l e G D W w a s a d d e d t o f u r t h e r d u p l i c a t e sa m p l e s ,

g iv in g a r a n g e o f m o i s t u r e c o n t e n t s b e t w e e n 3 0 a n d 9 0 .

T h e f l a sk s w e r e i n c u b a t e d f o r 3 d a y s a t 2 5 ° C o n a n

o r b i t a l s h a k e r ( L H E n g i n e e ri n g C o . , E n g l a n d ) . A f t e ri n c u b a t i o n , t h e c u l t u r e s w e r e ly o p h i l i z e d b e f o r e e s t i m a t i n gr e s id u a l p h y t a t e i n t h e t r e a t e d m e a l u s i ng a m o d i f i c a t i o n

o f t h e m e t h o d d e sc r ib e d b y U p p s t r o m a n d S v e n s o n.26

T r i p l ic a t e s a m p l e s ( 1 g ) o f l y o p h i l i z e d m e a l w e r e e x t r a c t e d

w i t h 2 0 m l 1 M h y d r o c h l o r i c a c i d f o r 1 h a t 2 5 ° C . A f t e r

c e n t r i f u g a t i o n , 6 m l o f t h e s u p e r n a t a n t w a s p i p e t t e d i n t o

a 2 5 m l v o l u m e t r i c f l a s k f o l lo w e d b y 4 m l 2 . 8 M s o d i u m

a c e t a t e b u f f e r , p H 8 . 5 , c o n t a i n i n g 0 .1 M m a g n e s i u m

s u l p h a te a n d , f i n a l l y , 5 0 m g w h e a t 6 - p h y t a s e ( S i g m a

C h e m i c a l C o . L t d ) d i s so l v e d i n 5 m l d e m i n e r a l i z e d w a t e r ,

b e f o r e m a k i n g u p t o v o l u m e w i t h w a t e r . B a c k g r o u n d

p h o s p h a t e w a s m e a s u r e d u s i n g 1 m l o f t h i s s o l u t i o n a n d

t h e r e m a i n d e r w a s i n c u b a t e d f o r 2 4 h a t 3 7 ° C , w h e n t h e

p h o s p h a t e w a s m e a s u r e d i n a s e c o n d a l i q u o t ( 1 m l ) .

P h o s p h a t e w a s m e a s u r e d u s in g t h e m e t h o d o f A l l e n 2 7

b y r e a c ti n g t h e s a m p l e w i t h a m m o n i u m m o l y b d a t e , a f t e r

a c i d i f ic a t i o n , an d m e a s u r i n g t h e r e s u l t a n t b l u e c o l o u r o n

a P y e- U n i c a m S P6 0 0 s p e c t r o p h o t o m e t e r a t 7 8 0 n m . T h e

c a l i b r a ti o n c u r v e w a s p l o t t e d f r o m KH PO 4 so lu t ions and

p h y t a t e p h o s p h o r u s e s t i m a t e d a s th e i n c r e a s e a ri s in g f r o m

e n z y m e h y d r o l y s i s .

I n i t ia l le v e ls o f p h y t a t e w e r e e s t i m a t e d o n u n t r e a t e d

s a m p l e s o f r a p e s e e d m e a l .

S p o r e s o r c e l ls o f a r a n g e o f m o u l d s a n d y e a s t s w e r e

g r o w n a n d h a r v e s t e d a s d e sc r i b e d f o r R. oligosporus. T h e

f i n a l sp o r e / c e l l s u sp e n s i o n w a s a d j u s te d t o ~ 1 0 7 m 1 - 1 a n d

u s e d t o i n o c u l a t e t h e f o l lo w i n g m e d i a .

( a ) R a p e s e e d m e a l . S a m p l e s w e r e s e t u p a s d e s c r i b e d f o r

R. oligosporus, w i t h a f i n a l m o i s t u r e c o n t e n t o f 9 0 .I n c u b a t i o n a n d p h y t a t e e s t i m a t i o n w a s c a r r ie d o u t a s

desc r ibed prev ious ly .

(b ) P hytase s c reen ing m edium (P S M) aga r . Th i s was a

s y n t h e t i c m e d i u m o f t h e f o l l o w i n g c o m p o s i t i o n ( g 1 -1 ) ;

D - g l uc o s e, 1 5 . 0 ; c a l c i u m p h y t a t e , 5 . 0 ; N H 4 N O a , 5 . 0 ;

M g S O 4 • 7 H 2 0 , 0 . 5 ; K C 1, 0 . 5 ; F e S O 4 • 7 H 2 0 , 0 . 0 1 ;

M n S O 4 • 4 H 2 0 , 0 . 01 ; m a d e t o v o l u m e w i t h d i st i ll e d w a t e r

a n d s o l i d if i e d w i t h D i f c o - B a c t o N e w Z e a l a n d a g a r . T h e p H

was ad jus ted to 5 .5 us ing 1 M HCI be fo re au toc lav ing a t

1 2 1 ° C f o r 2 0 m i n . D u p l i c a t e p l a t es w e r e i n o c u l a t e d w i t h

0 .1 m l o f t h e s p o r e / c e ll s u s p e n s io n , a d j u s t e d t o c o n t a i n

l 2 spores m 1-1 . The spread p la te s were then incuba ted a t

2 5 ° C f o r 4 d a y s , a f te r w h i c h t i m e g r o w t h a n d c l e a r a n c eof the aga r gave a v i sua l ind ica t ion o f ex t race l lu la r phy tase

p r o d u c t i o n ( s e e Figure 1 .( c ) P SM b r o t h . S a m p l e s ( 1 0 0 m l ) o f PS M b r o t h w e r e

inocu la ted w i th 0 .1 m l o f a suspens ion con ta in ing 107 spores /

c e ll m 1- 1 a n d i n c u b a t e d f o r 4 d a y s a t 2 5 ° C o n a n o r b i t a l

3 7 8 E n z y m e M i c r o b . T e c h n o l . , 1 9 8 3 , V o l . 5 , S e p t e m b e r



s h a k e r . T h e c u l t u r e w a s t h e n f i l t e r ed u s i n g W h a t m a n N o . 1

f i lt e r p a p e r a n d t h e f i l t r a t e c e n t r i f u g e d a t 1 2 0 0 0 r e v / m i n a t5 ° C f o r 3 0 m i n . T h e s u p e r n a t a n t s w e r e t h e n s t o r e d a t - - 2 0 ° C

p r i o r t o a s s ay i n g p h y t a s e a c t i v i t y .

I n t o t e s t t u b e s w e r e p i p e t t e d 0 . 5 m l o f 0 .2 M s o d i u m

a c e t a t e b u f f e r ( p H 4 . 4 ) , 0 . 5 m l s o d i u m p h y t a t e i n 0 . 2 M

s o d i u m a c e t a t e b u f f e r f o l l o w e d b y 1 m l o f c u l t u r e f i l t r a t e ,

g iv ing a f ina l pH o f 5 . 5 . Dupl ica te s am ples were incub a ted

i n a w a t e r b a t h a t 4 0 0 C f o r 0 a n d 4 5 m i n , b e f o r e t h e

r e a c t i o n w a s s t o p p e d b y a d d i n g 2 m l 1 5 T C A . O r t h o -

p h o s p h a t e i n t h e r e a c t i o n m i x t u r e w a s e s t i m a t e d a c c o r d i n g

t o A l l en 2 7 a n d p h y t a s e a c t i v i t y m e a s u r e d a s t h e a m o u n t o f

p h o s p h a t e r e l e a s ed d u r i n g t h e i n c u b a t i o n t i m e . O n e u n i t

o f p h y t a s e a c t i v i t y w a s d e f i n e d as t h e a m o u n t o f e n z y m e

c a p a b l e o f r e l e as i n g o n e m o l e o f i n o r g a n ic p h o s p h o r u s i n

o n e m i n u t e u n d e r t h e d e f i n e d r e a c t i o n c o n d i t i o n s .

( d ) D i f c o - B a c t o P o t a t o d e x t r o s e b r o t h ( P D B ) . I n o c u l a t i o n ,

i n c u b a t i o n , r e c o v e r y a n d p h y t a s e a s s a y o f c u l t u r e f i l t r a t e s

in P DB was ca r r i ed ou t a s fo r P S M bro th .

Ch a r a c te r iz a ti o n o f t h e p h y ta s e o fA s p e r g i l l u s f i c u u m

F l a s k s co n t a i n i n g 1 0 0 m l P S M b r o t h w e r e i n o c u l a t e d

w i t h 1 0 6 s p o r e s o f A . ficuum N R R L 3 1 3 5 a s d e s cr i be d

a b o v e , a n d i n c u b a t e d o n a n o r b i t a l s h a k e r a t 2 5 ° C . D u p l i-

c a t e c u l t u r e s w e r e h a r v e s te d d a i l y u p t o 1 6 d a y s , t h e

f i l t ra t e p u r i f i e d a n d a s s a y e d f o r p h y t a s e a c t i v i ty a s d e s c r ib e d .

T h e t e m p e r a t u r e p r o f i l e o f p h y t a s e a c t i v i t y i n P SM

c u l t u r e f i l t r a te s o f A . fieuum w a s d e t e r m i n e d b y a s s a y s o f

t r ip l i ca te s am ples a s desc r ibed p rev iou s ly ove r a range of

2 0 ° C t o 7 0 ° C . B l a n k s a n d te s t s w e r e b o t h p e r f o r m e d a t

e a c h t e m p e r a t u r e a f t e r e q u i l i b r a t i o n o f a l l c o m p o n e n t s

p r i o r t o t h e r e a c t i o n .

T h e p H p r o f i l e o f p h y t a s e a c t i v i t y w a s d e t e r m i n e d a t

4 0 ° C . T o a c h i e v e a w i d e p H r a n g e , t h e s o d i u m p h y t a t e

c o n c e n t r a t i o n w a s r e d u c e d t o 1 0 m g m 1 - 1 o f r e l e v a n t

b u f f e r . B u f f e r s u se d w e r e : S o r e n s e n s g l y c in e I ( p H 2 . 1 5 -

3 . 0 5 ); K o l th o f f s b o r a x - s u c c i n a t e ( p H 3 . 8 7 - 7 . 1 4 ) a n d

S o r e n s e n s c i t r a te I I ( p H 7 . 2 5 - 8 . 0 ) . 2 8 T h e a s s a y w a s

c o n d u c t e d u s in g d u p l i c a t e s a m p l e s a s d e s c r i b e d , a n d t h e

p H o f t h e f i n a l r e a c t i o n m i x t u r e w a s re c o r d e d .

T h e e f f e c t o f i n o r g a n i c p h o s p h a t e o n t h e d e v e l o p m e n t

o f p h y t a s e a c t i v i t y d u r i n g g r o w t h w a s t e st e d b y i n c u b a t i n g

d u p l i c a t e s a m p l e s o f A . fieuum a t 25°C for 4 days in P S M

b r o t h c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f K H 2 P O 4 a s

t h e o n l y s o u r c e o f i n o r g a n ic p h o s p h a t e Table 4).T h e e f f e c t o f v a r i a t io n o f t h e n i t r o g e n s o u r c e o n e n z y m e

p r o d u c t i o n w a s t e s t e d b y a l t e r i n g t h e n i t r o g e n o u s c o m -

p o u n d s p r e s e n t i n t h e b a s i c P SM b r o t h Table 5). The f ina l

l e ve l o f n i t r o g e n i n e a c h m e d i u m w a s t h e s a m e a s in t h e

bas ic P S M bro th a s desc r ibed ea r l i e r . The phytase a s say

w a s c a r r ie d o u t a s a l r e a d y d e s c r ib e d a f t e r a d j u s t m e n t o f t h e

p H o f t h e c u l t u r e f i l t r a te s t o 5 .5 u s i ng 0 .1 M N a O H o r

1 M HC1 as neces sa ry .

Co m p a r i s o n o f a c t i v i ty o f wh e a t 6 - p h y ta s e a n dA . f i c u u m p h y ta s e o n R S M

S i x l it r es o f P SM w a s i n o c u l a t e d w i t h 5 m l o f a s p o r e

s u s p e n s i o n o f A . fieuum conta in ing 107 spores m l -x . The

c u l t u r e w a s i n c u b a t e d a t 2 5 ° C o n a n o r b i t a l s h a k e r( 2 5 0 r e v ] m i n ) f o r 1 0 d a y s . I t w a s th e n f i l te r e d t h r o u g h

W h a t m a n N o . 1 fi l te r p a p e r a n d t h e s u p e r n a t a n t c e n t r i -

f u g ed a t 1 2 0 0 0 r e v / m i n fo r 3 0 m i n a t 4 ° C . T h e 4 9 0 0 m l

o f c u l t u r e f i l t r a te o b t a i n e d w a s ly o p h i l i z e d ( E d w a r d S h e l f

M o d e l E G 6 ( S ) ) a n d t h e r e s u l t a n t p r e p a r a t i o n w a s s u s p e n d e d

S t u d ie s o f m i c r o b i a l p h y t a s e : S J H o w s o n a n d R P D a v is

i n 4 0 m l o f g l a s s -d i s ti l le d w a t e r . T h i s w a s a s sa y ed a n d f o u n d

t o h a v e a n a c t i v i t y o f 0 . 6 3 4 U m 1 - 1.

T h is p h y t a s e w a s th e n c o m p a r e d w i t h a c o m m e r c i a l

p r e p a r a t i o n o f w h e a t 6 - p h y t a s e ( S ig m a C h e m i c a l C o . ) .

T h e w h e a t 6 - p h y t a s e h a d a n a c t i v i t y o f 0 . 0 1 8 U m g - 1 so l i d.

T o 5 g o f m e a l w a s a d d ed 5 0 0 m g o f o n e o f t h e p h y t a s e

p r e p a r a t i o n s a n d 4 0 m l o f g l a ss - di s ti ll e d w a t e r ( e q u i v a l e n t

m o i s t u r e c o n t e n t 9 0 ) . D u p l i c a t e sa m p l e s w e r e in c u b a t e d

in 100 m l con ica l f l a sks a t 40°C in a shak ing incu ba to r( N e w B r u n s w i c k L t d ) . T h e c o n d i t i o n s o f i n c u b a t i o n v a r i e d

a s f o l lo w s : ( a ) 2 4 h ; ( b ) 4 8 h ; ( c ) a f t e r 2 4 h a n o t h e r 5 0 0 m g

o f e n z y m e w a s a d d e d a n d i n c u b a t e d f o r a f u r t h e r 2 4 h ;

( d ) a s f o r (c ) e x c e p t 5 g m e a l w a s a d d e d r a t h e r t h a n t h e

e n z y m e .

A f t e r i n c u b a t i o n t h e s e s a m p l e s w e r e t h e n e x t r a c t e d a n d

as sayed a s desc r ibed prev ious ly .

R e s u l t s a n d d i s c u s s i o n

T h e o p t i m u m m o i s t u r e c o n t e n t f o r p h y t a s e a c t i v it y , a s

s h o w n b y t h e r e d u c t i o n o f p h y t a t e , i n r a p e s ee d m e a l in c u -

b a t e d f o r 3 d a y s a t 2 5 ° C a f t e r i n o c u l a t i o n w i t h s p o r e s o fR. oligosporusw a s 8 0 Table 1). Reinh old 29 a l so

r e p o r t e d i n c r e a s e d r e m o v a l o f p h y t a t e f r o m b r e a d d o u g h s

i n c u b a t e d w i t h Saccharom yces cerevisiaea t h i g h m o i s t u r e

c o n t e n t s . G l a ss a n d G e d d e s a ° f o u n d t h a t p h y t a s e a c t i v i ty

w a s n o t d e t e c t a b l e i n d r y d o r m a n t w h e a t g r a in s w h i ls t

ac t iv i ty inc reased wi th r i s ing m ois tu re con ten t dur ing

s to rage .

T h e r e d u c t i o n o f p h y t a t e i n r a p e s e e d m e a l a c h i e v e d b y

a r a n g e o f f u n g i a n d y e a s t s , a f t e r i n c u b a t i o n u n d e r s i m il a r

cond i t ions , va r ied be tw een 10 and 80 . A s im i la r degree

o f v a r i a t io n i n t h e r e d u c t i o n o f p h y t a t e w a s o b s e r v e d f o r

d i f fe ren t s t ra ins o f a g iven spec ies ; da ta fo r Aspergillus nigera re shown in Table 2. E x t r a c e l h i la r p h y t a s e a c t i v i ty w a sde tec ted in 33 ou t o f 84 s t ra ins t e s ted in P S M and in 37

s t ra ins in P DB Table 3). A l l s t ra i n s w h i c h s h o w e d e n z y m e

able 1 R e d u c t i o n o f p h y t a t e i n r a pe s e e d m e a l i n o c u l a t e d w i t h

c o n i d i a o f hizopus oligosporusa n d i n c u b a t e d f o r 3 d a y s at 3 0 ° C

P h y t a t e c o n t e n t

M o i s t u r e c o n t e n t o f m e a l( ) ( m g g - l ) r e d u c t i o n

2 5 3 6 . 0 1 8

3 0 3 7 . 2 1 5

4 0 3 8 .1 1 4

5 O 3 6 .8 1 66 0 3 5 . 0 2 0

7 0 3 1 . 2 2 9

8 0 1 6 . 0 6 4

9 0 2 0 . 5 5 4

ab le 2 Effec t of cul ture of various stra ins of A niger on thephyta te c on ten t o f rapeseed mea l

Res idua l phy ta tein meal Phytate reduc t ion

St ra in (mg g- l ) ( w/w )

L25 7.7 37.9CS180 8.2 34.3

L9 8.4 32.3CSl 4 4 .2 66 .0UD31 4 9 .7 21 .8LIO 10.9 12.2L26 10.9 12.1CS14 3 .8 69 .4

E n z y m e M i c r o b . T e c h n o l . , 1 9 8 3, V o l . 5 , S e p t e m b e r 3 7 9



P a p e r s

Table 3 S u r v e y o f m i c r o o r g a n i s m s f o r p h y t a t e h y d r o l y s i n g a c t i v i ty

i n c u l t u r e f i l t r a t e s

S t r a in s e x h i b i t i n g S t r a i n s e x h i b i t i n g

a c t i v i t y i n P S M a c t i v i t y i n P D B

P o s i t i ve Ne g a t i ve P o s i t i ve Ne g a t i ve

Asp e rg i l l u s n ig e r 0

A f i c u m 1

A p e t r a k i i 0

A c a n d i d u s 3

A f l a vu s 0

A w e n t i i 0

A v e r s i c o l o r 2

A s y d o w i 3

A re p e n s 2

A ch e va l ie r i 0

A a m s t e l o d a m i 3

S a c c h a r o m y c e s

cerevisiae 4

S u v a r u m 0

R h i z o p u s o r y z a e 2

R o l i g o s p o r u s 1

R s t o l o n i f e r 2

R a r r h i z u s 0

M u c o r r a c e m o s us 3

M p i r i f o r m i s 4

G e o t r i c h u m c a n d i d u m 1

B o t r y t i s c i n e r e a 2

C l a d os p o r i u m

c l a d o s p o r o i d e s 0

R h o d o t o r u l l a

g r a m i n i s 0

A o r y z a e 0

7 3 4

0 1 0

2 0 2

2 0 5

6 4 2

1 1 0

3 1 4

0 3 0

2 3 1

2 1 1

2 3 2

8 6 6

1 0 1

3 2 3

0 0 1

2 2 2

1 0 I

2 4 1

0 2 2

0 0 1

3 1 4

1 0 1

1 0 1

2 0 2

0 03

-~ 0 .0 2-g

>

~- o . o

T h e r e w a s l i t t l e c o r r e l a t i o n b e t w e e n p h y t a t e h y d r o l y s i n g

a c t i v i t y i n t h e t w o m e d i a F i gure 2 ) . M o r e o v e r , t h e r e w a s

l i t t l e c o r r e l a t i o n b e t w e e n t h e a b i l i t y to r e d u c e p h y t a t e i n

R S M a n d t o p r o d u c e a n e x t r a c e l l u l a r e n z y m e i n P S M o r

P D B F i gure s 3 a n d 4 .

T h e Aspergi l lus s p e c ie s a p p e a r e d t o b e t h e b e s t s o u r c e

o f e x t r a c e l l u l a r p h y t a s e e n z y m e s , w h i l s t A spe rg i ll u s f i c uu m

90

8 0

7 0

6 0

50 •

4 o •

3 t

A 4 L • A

t . & •

t I

0 0.01

I I I I I

0.07 O.07

PSM activity (units ml t)

Figure 3 R e d u c t i o n o f p h y t a t e c o n te n t o f R S M c o m p a r e d w i th

e x t r a c e l l u l a r p h y t a s e a c t i v i t y o f m o u l d s a n d y e a s t s w h e n g r o w n

in PSM

9O

8o

70

6 0

= = = •

• • ~ 50

0 0 . O I O . 0 2 O . 0 3 g 4 0

P D B a c t i v i t y ( u n i t s m l - I ) ~ •

Figure 2 E x t r a c e l l u l a r p h y t a s e a c t i v i t y o f v a r i o u s s t r a in s o f m o u l d s

a n d y e a s ts w h e n g r o w n i n P S M a n d P D B ~ 3 0

a c t i v i t y i n P S M b r o t h c u l t u r e s w e r e a l so s e e n to p r o d u c e z o ~ •

z o n e s o f c l e a r a n c e in a d v a n c e o f m y c e l i a l g r o w t h o n P S M

a g a r p l a t e s Figure 1) . T w e l v e s t r a i n s g a v e p h y t a s e a c t i v i t y •

i n b o t h P S M f i l t r a t e s a n d P D B , i n d i c a t i n g t h a t t h e e n z y m e to

w a s c o n s t i t u t i v e , w h i ls t 2 1 s t r a i n s s h o w e d p h y t a s e a c t i v i t y

o n l y i n PS M b r o t h i n d i c a t i n g t h a t t h e e n z y m e i s i n d u c e d b y L, jt h e p r e s e n c e o f t h e s u b s t r a t e . H o w e v e r , 2 5 s t r a in s t e s t e d o

s h o w e d a c t i v i t y o n l y i n P D B f i l t r a t e s a n d n o t i n P SM ,

w h i c h m a y h a v e b e e n a n e f f e c t o f s u b s t ra t e i n h i b i t i o n .

T h e t o t a l n u m b e r o f a c t i v e s t r a i n s w a s 5 8 ; 2 6 s t r a i n s

s h o w e d n o a c t i v i t y i n t h e c u l t u r e f i l t ra t e s .

| | I |

0 .0 4 0 .0 7

PDB activity un its ml l)

F igure 4 R e d u c t i o n o f p h y t a t e c o n t e n t o f R S M b y s t r a in s o f

m o u l d s a n d y e a st s w h e n c o m p a r e d t o t h e i r e x t r a c e l l u l a r p h y ta s e

a c t i v i t y i n P D B

3 8 0 E n z y m e M i c r o b . T e c h n o h , 1 9 8 3 , V o l . 5 , S e p t e m b e r



pr ov ide d the m o s t ac t ive cu l tu r e f i l t r a te s . S h ieh and W ar e 18

a l so f o u n d t h a t Aspergillus s p e ci e s c o m m o n l y p o s s e s s e d

p h y t a s e e n z y m e s a n d t h a t t h e m o s t a c t i v e p h y t a s e e x t r a c t s

w e r e p ro d u c e d b y Aspergil lus f icuum. B o t h i n t e r - a n d

in t r a spec ie s va r ia t ion was obse r ved .

P r o p e r t ie s o f e x t r ac e l lu l a r p h y ta s e f r o mA s p e r g i l l u s f i c u u m

The h ighes t ex t r ace l lu la r phy tase ac t iv i ty in P S M cu l tu r e

f i l tr a t e s o f A . f i e u u m w a s r e c o r d e d a f t e r 1 0 d a y s i n c u b a t i o n

a t 2 5 ° C (Figure 5), w h e n t h e a c t i v i t y o f t h e c u l t u r e f i l tr a t e

w a s a p p r o x i m a t e l y 6 t im e s g r e a t e r t h a n a t 4 d a y s .

S k o w r o n s k i 31 f o u n d t h a t p h y t a s e a c t i v i ty i n c u l t u r e f i l tr a t e s

o f t h e c l o s e ly r e l a t ed f u n g u s A. niger r e a c h e d a p e a k a f t e r

11 to 12 days . Th e s i tua t ion va r ie s wi t h spec ie s . S t r a ins o f

Ge o t r ie h u m e a n d i d u m s h o w e d o p t i m u m a c t iv i t y a t 4 d a y s

u n d e r t h e s a m e c o n d i t i o n s ; a f t e r t h is t i m e , a c t i v i t y w a s

r e d u ce d b y > 6 0 ( d a t a n o t s h o w n ) .

T h e i n c r e a s e in a c t i v i t y w i t h t i m e r e c o r d e d i n c u l t u r e

f i l tr a t e s o f A . f i e u u m c o u l d h a v e b e e n a r e su l t o f a u t o l y s i s

r a t h e r t h a n i n d u c t i o n , a s th e e n z y m e a p p e a r s t o b e c o n s t i -

t u t i v e . T h e m y c e l i a l m a t d i s i n te g r a t e d a f t e r 1 1 - 1 2 d a y s ,

i n d i c a ti n g t h a t a u t o l y s i s o f t h e m y c e l i u m o c c u r r e d a n d

c o u l d r e s u l t i n l e a k a g e o f i n t ra c e l lu l a r e n z y m e i n t o t h e

m e d i u m . S k o w r o n s k i 3 1 r e a c h e d a s i m i la r c o n c l u s i o n b y

e x c l u d i n g s u b s t r a t e i n d u c t i o n b y c o m p o n e n t s o f t h e

c u l t u r e m e d i u m .

T h e t e m p e r a t u r e o p t i m u m f o r p h y t a s e a c ti v i ty w a s 5 5 ° C ;

the ac t iv i ty a t 40°C, used f o r the s tanda r d ac t iv i ty a s say ,

w a s 4 0 o f t h a t at 5 5 ° C (Figure 6). S k o w r o n s k i 32 r e p o r t e d

a s i m il a r o p t i m u m ( 5 3 ° C ) f o r t h e c l o s e l y r e la t e d s p e c i e s

A. niger, w h i l e r e p o r t s f o r A . terreus give a r ange be tw een

60 and 70°C . 33 ' 34 Al l th r ee spec ie s a r e cons ide r ed to be

ve r y c lose ly r e la ted and a ll have been c la s s if i ed a s A. nigerin th e ~ast.35 S im i l a rl y h ig h o p t i m u m t e m p e r a t u r e s f o r

0 0 9

0 0 7

0 0 5

a o 3

0.OI

O 2 4 6 8 I 0 12 14 16

Age of cuture days)

F i g u r e 5 T i m e c o u r s e o f th e p r o d u c t i o n o f p h y t a t e h y d r o l y s i n g

e n z y m e i n t h e c u l t u r e f i l t r a t e o f s p e r g i ll u s f ic u u m N R R L 3 1 3 5

g r o w n i n P S M

S t u d i es o f m i c r o b i a l p h y t a s e : S J H o w s o n a n d R P D a v is

0.05

0 0 4

O 03

< 0.02

0.01

I I I I I I

35 40 45 50 55 60 65

Temperature *C)

F i g u r e 6 Temperature p r o f i l e o f t h e e n z y m e s c a p a b l e o f hydro-

lysing p h y t a t e in the culture filtrate of sperg i l lus f i cuum N R R L

3135 grown in PSM

70

8O

6O

_>

4O

2

I I = t I

2 3 4 5 6 7pH

F i g u r e 7 pH p r o f i l e o f t h e c u l t u r e f i lt r a t e o f Aspergillus ficuumN R R L 3 1 3 5 w i t h r e sp e c t t o p h y t a t e h y d ro l y s is

p h y t a s e a c t i v it y h av e b e e n r e p o r t e d f o r t h e e n z y m e f r o m

o t h e r m i c r o o r g a n i s m s 3 6 a n d f r o m p l a n t s .37

P h y t a s e a c t i v i t y s h o w e d t w o p H o p t i m a , o n e a t 2 . 0 a n d

a second le s s ac t ive peak a t 5 . 5 (Figure 7). P r e v i o u s r e p o r t s

h a v e a ls o s h o w n p h y t a s e e n z y m e s o f A . f i c u u m and r e la ted

Aspergillus s p e ci e s t o h a v e t w o p H op tim a. 19 31 32 3S S hiehet al. 3 8 s e p a ra t e d t h e t w o p e a k s b y c o l u m n c h r o m a t o g r a p h y

a n d t e r m e d t h e p H 2 . 0 f r a c t i o n a s a n o n - s p e c i fi c a ci d p h o s -

p h a t a s e a n d t h e p H 5 .5 f r a c t i o n a s p h y t a s e , w h i c h s t i ll

s h o w e d a c t i v i t y a t p H 2 . 0 , a l t h o u g h a t a l o w l e v e l.

S k o w r o n s k i 31 s h o w e d t h a t t h e p e a k s r e p r e s e n t e d t w o

E n z y m e M i c r o b . T e c h n o l . 1 9 8 3 V o l . 5 S e p t e m b e r 3 8 1



Papers

T a b l e 4 E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f i n o r g a n i c p h o s p h a t e o n

e x t r a c e l lu l a r p h y ta s e a c t i v i ty o f A ficuum in PSM

In o rg a n ic p h o s p h a t e c o n c e n t r a t i o n A c t i v i t y

(mg d l -~ ) (u n i t s m l -~ )

2 0 .2 4

5 0 .2 0

1 0 0 . 1 2

1 5 0 . 0 5

2 0 0 . 0 2

2 5 0

5 0 0

T a b l e 5 E f f e c t o f v a r i o u s n i t r o g e n so u rc e s o n e x t r a c e l l u l a r p h y t a s e

p r o d u c t i o n i n A . ficuum

A c t i v i t y R e l a t i v e a c t i v i t yN i t ro g e n so u rce (u n i t s m l - l ) ( ) P S M

( N H 4 ) 2 S O 4 0 . 0 2 6 1 7 3 . 3

U r e a 0 . 0 0 4 2 6 . 7

N H 4 H 2 P O 4 0 . 0 2 6 1 7 3 . 3

N a N O 3 0 . 0 1 2 8 0 . 0

K N O 3 0 . 0 1 4 9 3 . 4

N H , N O 3 ( s t a n d a r d P S M ) 0 . 0 1 5 1 0 0

T a b l e 6 C o m p a r i s o n o f r e d u c t i o n o f p h y t a t e i n R S M b y w h e a t

6 - p h y t a s e a n d A ficuum p h y t a s e u n d e r d i f f e r e n t c o n d i t i o n s

( 5 0 0 m g e n z y m e + 4 0 m l G D W + 5 g m e a l i n c u ba t e d a t 4 0 ° C )

C o n d i t i o n s r e d u c t i o n

W h e a t 6 p h y t a s e

2 4 h 7 3 .4

4 8 h 8 0 . 3

2 4 h + 5 0 0 m g e n z y m e 8 7 . 0

2 4 h + 5 0 0 m g m e a l 7 3 . 0

A ficuum p h y t a s e

2 4 h 6 5 . 2

4 8 h 6 5 . 9

2 4 h + 5 0 0 m g e n z y m e 8 4 . 0

2 4 h + 5 0 0 mg me a l 8 0 .2

enzymes as they exhibited markedly different heat stability

propert ies. In con tras t, Irving and Cosgrove 19 fou nd it

impossible to discern whether the two peaks represented

one or two enzymes as they failed to separate the two

active centres either b y gel electrophoresis or the

visualization method .

Shieh e t a l . 38 showed that increased levels of inorganic

phosphorus in the growth medium suppressed synthesis of

both enzy mes although the ratio of the pH 2.0 to the

pH 5.0 peaks was reversed, and the phytase enzymebecame the most active enzy me produced. This was con-

firmed by our experime nts in which differe nt levels of

inorganic phospho rus were added to PSM brot h, which

contains negligible inorganic phosphorus. Inhi biti on of

enzyme pr oduc tion occurre d above 0.2 mg m1-1 of inorganic

phosphorus , as can be seen from the results in T a b l e 4 .

Phytate production is also influenced by nitrogen

source, with ammonium ions being markedly superior to

nitrate and urea ( T a b l e 5 ) . We found that urea supported

little growth. Ammo niu m salts were also foun d to be the

best nitrogen source in the medium for subsequent phytase

production by A . t e r r e u s cultures.34

It was of interest to compare the activity towardsphytate in RSM of phytate degrading enzymes from

A . f i c u u m and from wheat which is commercially available.

Both enzymes decreased the phytic acid content to a sub-

stantial degree, with the p reparat ion from wheat being

somewhat more active.Residual phytate was present in

RSM even after prolonged in cubat ion. This result does not

reflect loss of activity by the en zyme for the additio n to

the i ncubation mixture containing enzyme from either

source of more RSM led to fu rther removal of phytate

( T a b l e 6 ) . It may be that some proportion of the phytate

in RSM is inaccessible to the enzy me, perhaps by its

occlusion within cells or by cellular const ituent s. The

addition of further enzyme to meal in which phytate had

already been substa ntially degraded by earlier enzyme

addition did not result in complete breakdown of phytate

(data not shown).

e f e r e n c e s

1 Webster, . E.J. Agr ic . Re s . 1928, 37,12 3-12 52 Oberleas,D. and Harland, B. F.J. A m . D i e t . A s s o c . 1981,

79,433-4363 Erdman, J. W. J. Am. Oi l . Che m. Soc . 1979, 56,73 6-7414 Cheryan, M. C R C C n t . R e v . F o o d S cL N u t r . 1980, 13,

297-3355 Cosgrove, D. J. R e v . P u r e A p p l . C h e m . 1966, 16,2096 Asada,K., Tanaka, K. and Kasai, Z . P l a n t C e l l P h y s i o l . 1968,

9,1957 Fontaine, T. D., Pens, W. A. and Irving, G. W. J. Biol . Che m.

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