Enzymes in Food and Beverage Processing

7 Immobilized Enzymes A L F R E D C. OLSON and ROGER A. KORUS

Downloaded by UNIV LAVAL on March 4, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch007

Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, Calif. 94710

A n i m m o b i l i z e d e n z y m e has b e e n d e f i n e d as an e n z y m e that i s c o n s t r a i n e d one way o r a n o t h e r w i t h i n the l i m i t e d c o n f i n e s of a s o l i d support. T h e subject of i m m o b i l i z e d e n z y m e s has r e c e i v e d a s i g n i f i c a n t a m o u n t of attention i n r e c e n t y e a r s b e c a u s e of a d v a n t a g e s that h a v e b e e n and m i g h t be a c h i e v e d through s u c c e s s f u l a p p l i c a t i o n s i n a n u m b e r of f i e l d s i n c l u d i n g food p r o c e s s i n g . T h i s c h a p t e r on i m m o b i l i z e d e n z y m e s h a s b e e n divided into f i v e p a r t s b e g i n n i n g with an i n t r o d u c t i o n in w h i c h we h a v e t r i e d to p o s i t i o n i m m o b i l i z e d e n z y m e s w i t h i n the larger context of the use of e n z y m e s i n food p r o c e s s i n g , p o i n t i n g out a d v a n t a g e s and l i m i t a t i o n s to the method. T h i s i s f o l l o w e d by a s e c t i o n on s u p p o r t s that h a v e b e e n u s e d to i m m o b i l i z e e n z y m e s p a r t i c u l a r l y f o r food a p p l i c a t i o n s . A t h i r d s e c t i o n c o v e r s s o m e e x a m p l e s of i m m o b i l i z e d e n z y m e s c u r r e n t l y i n u s e i n food p r o c e s s i n g f o l l o w e d by a s e c t i o n on p r o p o s e d a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s . S i n c e m u c h of the l i t e r a t u r e on the s u b j e c t e x p r e s s e s r e s u l t s i n t e r m s that m a k e c o m p a r i s o n between d i f f e r e n t m e t h o d s of i m m o b i l i z a t i o n d i f f i c u l t , the f i n a l s e c t i o n i s a b r i e f d i s c u s s i o n of s o m e of the c o m m o n p a r a m e t e r s u s e d to d e s c r i b e i m m o b i l i z e d e n z y m e s y s t e m s . The c h a p t e r i s a s e l e c t e d s u r v e y of the r e c e n t l i t e r a t u r e with s o m e a p p l i c a t i o n s c o v e r e d i n m o r e d e t a i l i n o t h e r c h a p t e r s i n this book. T h e i n genuity, c o m p l e x i t y and d i v e r s i t y of m e t h o d s f o r i m m o b i l i z i n g e n z y m e s has b e e n l o o k e d at from the point of v i e w of t h e i r app l i c a t i o n to food p r o c e s s i n g , with s o m e s u g g e s t i o n s as to the f u t u r e u s e s of i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g . A s l i t t l e as 20-30 y e a r s ago a g r e a t d e a l of a r t and t r a d i t i o n w e r e still i n v o l v e d i n the use of e n z y m e s to p r o c e s s foods. M u c h of the t e c h n i c a l knowledge we now h a v e r e g a r d i n g e n z y m e s and t h e i r a p p l i c a t i o n to food p r o c e s s i n g has b e e n d i s c o v e r e d and d e v e l o p e d i n the i n t e r v e n i n g y e a r s . B a s i c i n f o r m a t i o n on e n z y m e s r e l a t e d to food c a n be found i n b o o k s b y R e e d (1) and W h i t a k e r (2,_3). M o r e s p e c i a l i z e d b o o k s by Z a b o r s k y (4), P y e and W i n g a r d (5), D u n l a p (6), O l s o n and C o o n e y (7) and S a l m o n a , S a r o n i o and G a r a t t i n i (8) d i s c u s s the u s e of i m m o b i l i z e d

100 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


7.

OLSON

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101

Immobilized Enzymes

e n z y m e s and r e l a t e d s u b j e c t s . A n u m b e r of r e v i e w s on i m m o b i l i z e d e n z y m e s h a v e a l s o a p p e a r e d (9-15). W i t h the s i t u a t i o n c h a n g i n g so r a p i d l y the p r o b l e m of des c r i b i n g new d e v e l o p m e n t s i n a p p l i e d food e n z y m o l o g y b e c o m e s v e r y d i f f i c u l t . T h e r e i s often a d i c h o t o m y of i n t e r e s t between b a s i c s c i e n t i f i c studies and c o m m e r c i a l a p p l i c a t i o n s . Ideas, e x p e r i m e n t s , p r o c e s s e s and p r o d u c t s that l o o k good i n the l a b o r a t o r y m a y not m e e t m a r k e t i n g r e q u i r e m e n t s , c u s t o m e r d e s i r e s or the i n t e r e s t of a p r o m o t e r . A l a r g e i n v e s t m e n t by a c o m p a n y in a new p r o c e s s i n v o l v i n g e n z y m e s w i l l p r o b a b l y r e s u l t i n the c o m p a n y k e e p i n g that i n f o r m a t i o n p r o p r i e t a r y a s l o n g a s p o s s i ble. T o p r o j e c t what r o l e e n z y m e s , i n c l u d i n g i m m o b i l i z e d enz y m e s , m a y h a v e i n food p r o c e s s i n g i n the f u t u r e i s even m o r e d i f f i c u l t . T h e e x e r c i s e i s not without m e r i t , h o w e v e r , f o r an o v e r v i e w of the s i t u a t i o n often c l a r i f i e s o b j e c t i v e s and b r i n g s into f o c u s a r e a s w h e r e p r o g r e s s can be m a d e . A study of r e c e n t d e v e l o p m e n t s i n the use of e n z y m e s i n food p r o c e s s i n g can be d i v i d e d into t h r e e a r e a s of i n v e s t i g a t i o n : e n z y m e p r o d u c t i o n , e n z y m e p u r i f i c a t i o n and e n z y m e a p p l i c a t i o n . A l l t h r e e a r e a s a r e of i m p o r t a n c e and p r o m i s e and a r e c u r r e n t l y r e c e i v i n g a g r e a t d e a l of attention. F o r e x a m p l e , by a p p l y i n g knowledge f r o m studies m a d e i n m i c r o b i a l g e n e t i c s and i n d u c e d e n z y m e s y n t h e s i s it m a y be p o s s i b l e to p r e p a r e m u c h l a r g e r a m o u n t s of i n t r a - and e x t r a - c e l l u l a r food grade e n z y m e s than was p o s s i b l e b e f o r e . T h e s e e n z y m e s can be u s e d as h e a t s t a b i l i z e d whole c e l l s o r p u r i f i e d b y a v a r i e t y of p r o c e d u r e s . A s u m m a r y of the steps i n v o l v e d i n the c o m m e r c i a l p r o d u c t i o n and p u r i f i c a t i o n of e n z y m e s f o r food use i s shown i n F i g u r e 1.

ENZYME

SOURCES

ENZYME

PURIFICATION

ANIMAL GLANDS EVAPORATOR

CENTRIFUGE OR FILTER PRESS

PLANT TISSUE

MEMBRANE FILTRATION

REVERSE OSMOSIS

CHROMATOGRAPHY

ACETONE PRECIPITATION

ELECTROPHORESIS SPRAY DRIER FRACTIONAL PRECIPITATION

FREEZE DRYER

~ T ~ MICROORGANISMS r

I

DRIER

I

DRY CRUDE PRODUCT FERMENTOR

CONCENTRATED LIQUID PRODUCT

FRACTIONATED PRODUCT

STANDARDIZED DRY PRODUCT

DILUTE LIQUID PRODUCT

Figure 1. Enzyme production and purification

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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PROCESSING

W h i l e s o m e e n z y m e s s u c h a s r e n n i n and p a p a i n a r e obtained f r o m a n i m a l and plant s o u r c e s , the m a j o r i t y a r e f r o m m i c r o o r g a n i s m s . P u r i f i c a t i o n i n v o l v e s s o m e k i n d of c o l l e c t i o n f o l l o w e d by a c o n c e n t r a t i o n step. E n z y m e p r e p a r a t i o n and purification that i s done i n b a t c h e s m a k e s r e p r o d u c i b i l i t y d i f f i c u l t and c o s t s h i g h . E f f o r t s a r e u n d e r w a y i n a n u m b e r of l a b o r a t o r i e s to be a b l e to c o n t i n u o u s l y p r o d u c e , p u r i f y , and u s e e n z y m e s . In the c o m m e r c i a l a p p l i c a t i o n of e n z y m e s it s h o u l d be advantageous to c o m b i n e continuous p r o d u c t i o n , p u r i f i c a t i o n and u s e of e n z y m e s , i n c l u d i n g i m m o b i l i z e d e n z y m e s , at a s i n g l e l o c a t i o n . T h e a b i l i t y to i m m o b i l i z e e n z y m e s , then, r e p r e s e n t s only one p a r t of a l a r g e r r e s e a r c h effort that i s c h a n g i n g food p r o c e s s i n g . What a r e s o m e of the advantages to i m m o b i l i z e an enzyme? B r i e f l y , they i n c l u d e (1) the a b i l i t y to u s e the e n z y m e m a n y t i m e s m o r e than the s o l u b l e e n z y m e can be u s e d , (2) l o w e r e n z y m e c o s t s (because of 1), (3) no e n z y m e left i n the p r o d u c t , (4) i n c r e a s e d s t a b i l i t y of the e n z y m e ( i m m o b i l i z a t i o n h a s b e e n shown to s t a b i l i z e s o m e e n z y m e s and p r o l o n g t h e i r a c t i v i t y on e x p o s u r e to s u b s t r a t e ) , (5) i m p r o v e d e n z y m e b e h a v i o r ( p H o p t i m a shifted to m o r e advantageous p H on i m m o b i l i z a t i o n to c e r t a i n s u p p o r t s ) , (6) continuous p r o c e s s p o s s i b l e , (7) b e t t e r q u a l i t y c o n t r o l of the p r o d u c t (because of 6), (8) l e s s a d d i t i o n a l p r o c e s s i n g (because of 3), (9) l o w e r l a b o r c o s t s , and (10) a d vantageous u s e of m u l t i p l e e n z y m e s y s t e m s . There are also p o t e n t i a l d i s a d v a n t a g e s w h i c h i n c l u d e (1) the cost of the i m m o b i l i z a t i o n , (2) l o s s of e n z y m e a c t i v i t y on i m m o b i l i z a t i o n , (3) g r e a t e r i n i t i a l plant i n v e s t m e n t , (4) m o r e t e c h n i c a l l y c o m p l e x p r o c e s s , (5) m o r e s k i l l e d s u p e r v i s i o n of p r o c e s s r e q u i r e d (because of 4), (6) h i g h e r l a b o r c o s t s (because of 5), (7) unique s a n i t a t i o n a n d t o x i c o l o g y p r o b l e m s , and (8) a p p l i c a b l e m a i n l y to soluble substrates. W h i l e these g e n e r a l i z a t i o n s c a n p r o v i d e s o m e g u i d e l i n e s , f i n a l c o n s i d e r a t i o n s that d e t e r m i n e w h e t h e r it is advantageous to i m m o b i l i z e an e n z y m e depend on the p a r t i c u l a r p r o c e s s one i s c o n s i d e r i n g . E n z y m e Supports In this s e c t i o n we h a v e c o v e r e d i n f o r m a t i o n on e n z y m e i m m o b i l i z a t i o n i n t e r m s of s u p p o r t s that h a v e b e e n u s e d f o r this purpose including polysaccharides, inorganic supports, fibrous p r o t e i n s , s y n t h e t i c p o l y m e r s , h y d r o g e l s and h o l l o w f i b e r s . E n z y m e s c a n be c o n s t r a i n e d o r h e l d to these s u p p o r t s b y a d sorption, covalent attachment, c r o s s l i n k i n g , entrapment, m i c r o e n c a p s u l a t i o n o r b y v a r i o u s c o m b i n a t i o n s of the f o r e g o i n g m e t h o d s . E x a m p l e s of these m e t h o d s of c o n s t r a i n t w i l l be d i s c u s s e d w h e r e they h a v e b e e n u s e d w i t h p a r t i c u l a r s u p p o r t m a t e rials. T h e v a r i e t y of s u p p o r t m a t e r i a l s that h a v e b e e n p r o p o s e d with s o m e s u i t a b i l i t y f o r a food p r o c e s s i n g a p p l i c a t i o n i s worth noting.



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Immobilized Enzymes

103

P o l y s a c c h a r i d e S u p p o r t s . A l a r g e n u m b e r of p o l y s a c c h a r i d e and substituted p o l y s a c c h a r i d e m a t e r i a l s have b e e n u s e d as s u p p o r t s for e n z y m e i m m o b i l i z a t i o n . T h e s e s u p p o r t s h a v e b e e n u s e d f o r a d s o r p t i o n of e n z y m e s i n a n u m b e r of food p r o c e s s i n g a p p l i c a t i o n s s u c h as p r o d u c t i o n of L - a m i n o a c i d s i n J a p a n w h i c h u s e s D E A E - S e p h a d e x (16, 17) and the C l i n t o n C o r n P r o c e s s i n g C o m p a n y p r o c e s s for h i g h f r u c t o s e c o r n s y r u p i n w h i c h g l u c o s e i s o m e r a s e a d s o r b e d on D E A E - c e l l u l o s e i s u s e d i n r e a c t o r s c o n s i s t i n g of a n a s s e m b l y of s h a l l o w beds of the i m m o b i l i z e d e n z y m e (18). C e l l u l o s e i s the m a j o r constituent of f i b r o u s plants and the m o s t abundant o r g a n i c m a t e r i a l i n n a t u r e . T h e m o n o m e r units of c e l l u l o s e a r e D - g l u c o s e . A l a r g e n u m b e r of substituted c e l l u l o s e s have b e e n p r e p a r e d by r e p l a c i n g the h y d r o x y l g r o u p s b y v a r i o u s substituents. R e p l a c e m e n t by d i e t h y l a m i n o e t h y l ( D E A E ) substituents i n t r o d u c e s a n a m i n o g r o u p w h i c h p r o v i d e s a p o l y c a t i o n i c , w e a k l y b a s i c a n i o n exchange g r o u p . D E A E c e l l u l o s e h a s b e e n one of the m o s t w i d e l y u s e d s u p p o r t s f o r e n z y m e i m m o b i l i z a t i o n . O n the other h a n d , r e p l a c e m e n t of h y d r o x y l g r o u p s by c a r b o x y m e t h y l ( C M ) substituents i n t r o d u c e s c a r b o x y l g r o u p s w h i c h p r o v i d e a w e a k l y a c i d i c c a t i o n exchange material. O t h e r p o p u l a r s u p p o r t s for e n z y m e i m m o b i l i z a t i o n a r e Sephadex and substituted Sephadex. Sephadex i s a b e a d e d , c r o s s l i n k e d d e x t r a n . D e x t r a n s a r e p o l y s a c c h a r i d e s of g l u c o s e units that a r e p r o d u c e d by s t r a i n s of L e u c o n o s t o c mesenteroides. Substituted Sephadex c a n be p r e p a r e d i n m u c h the s a m e way a s substituted c e l l u l o s e and shows a s i m i l a r ability for enzyme a d s o r p t i o n . E n z y m e s h a v i n g a r e l a t i v e l y h i g h content of a c i d i c a m i n o a c i d s r e m a i n f i r m l y bound to D E A E - c e l l u l o s e o r D E A E Sephadex even at h i g h s u b s t r a t e c o n c e n t r a t i o n a s l o n g as c e r t a i n p h y s i c a l c o n d i t i o n s s u c h as i o n i c s t r e n g t h and p H a r e m a i n t a i n e d . T h e a m i n o a c y l a s e f r o m A s p e r g i l l u s o r y z a e a d s o r b e d on D E A E - S e p h a d e x l o s e s 40% of its a c t i v i t y o v e r a 3 2 - d a y p e r i o d when u s e d at 5 0 ° C f o r continuous h y d r o l y s i s of a c e t y l a t e d L m e t h i o n i n e ( 19). D e t e r i o r a t e d c o l u m n s c o u l d be r e a c t i v a t e d b y d i r e c t a d d i t i o n of a m i n o a c y l a s e . H i g h e r e n z y m e a c t i v i t i e s w e r e obtained with D E A E - S e p h a d e x than with D E A E - c e l l u l o s e . U p w a r d flow i n a p a c k e d c o l u m n was u s e d to a v o i d c o m p r e s s i o n of the p a c k e d bed and e x c e s s i v e p r e s s u r e b u i l d - u p . T h e o p t i m u m c o n d i t i o n s of p H , i o n i c s t r e n g t h and t e m p e r a t u r e m u s t be d e t e r m i n e d to a c h i e v e and m a i n t a i n good a d s o r p t i o n and good a c t i v i t y . If a s t r o n g a d s o r p t i o n does not o c c u r , the e n z y m e i s r e a d i l y d e s o r b e d with subsequent l o s s of a c t i v i t y and c o n t a m i n a t i o n of the product. T h e s e l e c t i o n of o p t i m u m c o n d i t i o n s h a s m o s t l y b e e n a m a t t e r of t r i a l and e r r o r . M i c r o c r y s t a l l i n e D E A E - c e l l u l o s e powder is easily c o m p r e s s e d . O n l y s h a l l o w p a c k e d beds c a n be u s e d i n a l a r g e s c a l e c o m m e r c i a l o p e r a t i o n to a v o i d e x c e s s i v e p r e s s u r e b u i l d - u p .


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T h i s f l o w b e h a v i o r can be i m p r o v e d by u s i n g p o r o u s D E A E c e l l u l o s e beads w h i c h show a h i g h e n z y m e l o a d i n g c a p a c i t y f o r i m m o b i l i z a t i o n of g l u c o s e i s o m e r a s e (20). G l u c o s e i s o m e r a s e i m m o b i l i z e d by a d s o r p t i o n on D E A E - c e l l u l o s e was found to be s o m e w h a t l e s s stable than the f r e e e n z y m e a t 6 0 C . At50°C the i m m o b i l i z e d g l u c o s e i s o m e r a s e has a h a l f - l i f e of 10-11 d a y s u n d e r continuous o p e r a t i o n (21). A n o t h e r p r o m i s i n g p o l y s a c c h a r i d e support i s chitin. Chitin f o r m s the s t r u c t u r a l m a t e r i a l of the s k e l e t o n s of m a r i n e c r u s t a c e a n s and b e a r s the s a m e r e l a t i o n to i n v e r t e b r a t e s as c e l l u l o s e d o e s to p l a n t s . T h e m o n o m e r unit i s g l u c o s a m i n e . On about f i v e out of e v e r y s i x g l u c o s a m i n e u n i t s the a m i n o n i t r o g e n i s b l o c k e d with an a c e t y l group. N a t i v e c h i t i n contains a p p r o x i m a t e l y equal a m o u n t s of c a l c i u m c a r b o n a t e and c h i t i n . The c a l c i u m c a r b o n a t e can be r e m o v e d by a c i d g i v i n g a r i g i d p o r o u s m a t e r i a l that i s r e l a t i v e l y i n e r t to both c h e m i c a l and m i c r o b i a l attack. G l u c o s e i s o m e r a s e (22) and g l u c o a m y l a s e (23) h a v e b e e n i m m o b i l i z e d on c h i t i n by s i m p l e a d s o r p t i o n . T h e f o l l o w i n g e n z y m e s w e r e i m m o b i l i z e d on c h i t i n with g l u t a r a l d e h y d e : l a c t a s e ( a c i d t o l e r a n t ) , a c i d p h o s p h a t a s e , α-chymotrypsin (24) and g l u c o a m y l a s e (23). W h e n c h i t i n i s heated u n d e r p r e s s u r e with s t r o n g a l k a l i , i t is d e a c e t y l a t e d to p r o d u c e c h i t o s a n . T y p i c a l l y 8 0 % of the a c e t y l groups in chitin a r e r e m o v e d l e a v i n g f r e e amino groups in c h i t o s a n . C h i t o s a n has a l s o b e e n u s e d with g l u t a r a l d e h y d e to p r e p a r e i m m o b i l i z e d a c i d - t o l e r a n t l a c t a s e (25). T h e s e p o l y s a c c h a r i d e s a r e r e l a t i v e l y cheap, food g r a d e ma t e r i a l s and s o m e h a v e a h i g h c a p a c i t y f o r p r o t e i n a d s o r p t i o n . A s a g r o u p they a r e c u r r e n t l y a m o n g the m o s t w i d e l y u s e d sup p o r t s f o r e n z y m e i m m o b i l i z a t i o n i n the food i n d u s t r y .


e

I n o r g a n i c Supports. Porous glass, alumina, hydroxya p a t i t e , n i c k e l oxide on n i c k e l s c r e e n , s i l i c a a l u m i n a i m p r e g nated with n i c k e l oxide, p o r o u s c e r a m i c s of v a r i o u s types, s t a i n l e s s s t e e l and sand a r e s o m e of the i n o r g a n i c m a t e r i a l s to w h i c h e n z y m e s h a v e b e e n attached. A p p a r e n t a d v a n t a g e s of i n o r g a n i c s u p p o r t s f o r e n z y m e s i n c l u d e : (1) s t r u c t u r a l s t a b i l i t y o v e r a wide r a n g e of pH, p r e s s u r e , t e m p e r a t u r e , and s o l v e n t c o m p o s i t i o n , (2) e x c e l l e n t f l o w p r o p e r t i e s i n r e a c t o r s , (3) i n e r t n e s s to m i c r o b i a l a t t a c k or a t t a c k by e n z y m e s , (4) e a s e of a d a p t a b i l i t y to v a r i o u s p a r t i c l e shapes and s i z e s , and (5) good regeneration capability. A t t a c h m e n t of e n z y m e to these s u p p o r t s has b e e n by a d s o r p tion and by c o v a l e n t a t t a c h m e n t to the i n o r g a n i c s u r f a c e . S i n c e W e e t a l l (26, 27) f i r s t r e p o r t e d the u s e of p o r o u s g l a s s to i m m o b i l i z e e n z y m e s , m a n y e n z y m e s h a v e b e e n c o v a l e n t l y a t t a c h e d to m o d i f i e d p o r o u s g l a s s and r e l a t e d m a t e r i a l s (4, 28, 29). P o r o u s g l a s s c o m m o n l y u s e d f o r this p u r p o s e has p o r e d i a m e t e r s i n the range of 200-1500 A and p a r t i c l e s i z e i n the range of 20-80 m e s h . It i s 9 6 % s i l i c a and can be r e a c t e d with an a l k y l a m i n o


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Immobilized Enzymes

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E n z y m e s c a n then be c o u p l e d to the a m i n e d e r i v a t i v e by the c a r bodiimide, thiourea or a z o linkage methods which probably i n v o l v e a t t a c h m e n t to c a r b o x y l , a m i n e o r a r o m a t i c r e s i d u e s , r e s p e c t i v e l y , on the e n z y m e . A n a l t e r n a t i v e p r o c e d u r e i s to t r e a t the a l k y l a m i n e g l a s s w i t h g l u t a r a l d e h y d e f o l l o w e d b y the e n z y m e to effect a s t a b l e c o u p l i n g . P o r o u s g l a s s h a s b e e n r e p o r t e d to be p a r t l y s o l u b l e a n d b r e a k s down at p H v a l u e s n e a r 7 o r above. T h i s p r o b l e m c a n be o v e r c o m e by c o a t i n g the g l a s s w i t h z i r c o n i u m oxide, a v e r y i n s o l u b l e m a t e r i a l , p r i o r to u s e (30). Sand i s a n o t h e r s i l i c a s u p p o r t that h a s r e c e n t l y b e e n u s e d s u c c e s s f u l l y to i m m o b i l i z e β- gala c to s i d a se (31), a l c o h o l dehy d r o g e n a s e a n d u r e a s e (32). W h i l e sand h a s a l o w e r e f f e c t i v e s u r f a c e a r e a a n d e n z y m e l o a d i n g c a p a c i t y than p o r o u s g l a s s , i t i s l e s s e x p e n s i v e and thus m a y be s u i t a b l e f o r l a r g e v o l u m e a p p l i c a t i o n s w i t h l o w c o n c e n t r a t i o n s of s u b s t r a t e . A p p l i c a t i o n of the e n z y m e to sand c a n be a c c o m p l i s h e d b y p r e p a r i n g a l k y l a m i n e sand, t r e a t i n g this with g l u t a r a l d e h y d e f o l l o w e d b y the enzyme. P o r o u s c e r a m i c c a r r i e r s h a v e a l s o b e e n d e v e l o p e d and u s e d s u c c e s s f u l l y to b i n d e n z y m e s . T h e s e s u p p o r t s a r e l e s s e x p e n s i v e than p o r o u s g l a s s and m o r e i n s o l u b l e . A m i n o a c y l a s e h a s b e e n i m m o b i l i z e d on c e r a m i c c a r r i e r s c o m p o s e d of S i O , A l ^ O ^ , and T i O (33). C o u p l i n g was a g a i n a c h i e v e d by f i r s t p r e p a r i n g the s i i a n i z e d c e r a m i c s f o l l o w e d by t r e a t i n g w i t h g l u t a r a l d e h y d e and f i n a l l y a d d i n g e n z y m e . S o m e a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s i n v o l v e the s i m ple a d s o r p t i o n of e n z y m e s . F o r e x a m p l e , a p o r o u s c e r a m i c i s u s e d a s a n e n z y m e s u p p o r t i n the p r o d u c t i o n of 5'-mononucleo tide f l a v o r e n h a n c e r s (34). G l u c o s e i s o m e r a s e h a s b e e n s u c c e s s f u l l y a d s o r b e d on the i n t e r n a l s u r f a c e of c o n t r o l l e d p o r e a l u m i n a (35). T h e a l u m i n a was p r e t r e a t e d w i t h a d i l u t e s o l u t i o n of m a g n e s i u m a c e t a t e a n d cobalt a c e t a t e p r i o r to a d d i n g the



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enzyme. The resulting adsorbed glucose i s o m e r a s e exhibited c o n v e r s i o n s i n the r a n g e of 85% w i t h a h a l f l i f e i n c o l u m n o p e r a t i o n of 40 d a y s . G l u c o s e o x i d a s e has b e e n c o v a l e n t l y c o u p l e d to n i c k e l oxide on n i c k e l s c r e e n s (36) and to n i c k e l oxide i m p r e g n a t e d s i l i c a a l u m i n a p e l l e t s (37). M o r e r e c e n t l y M a r k e y , G r e e n f i e l d and K i t t r e l l (38) studied the i m m o b i l i z a t i o n of g l u c o s e o x i d a s e a n d c a t a l a s e on a n u m b e r of s i l a n i z e d i n o r g a n i c s u p p o r t s u s i n g g l u t a r a l d e h y d e as a c r o s s l i n k i n g agent. A d e c r e a s e i n a c t i v i t y of both e n z y m e s was o b s e r v e d as p a r t i c l e s i z e of the s u p p o r t s i n c r e a s e d up to 1000 m i c r o n s . T h i s d e c r e a s e was a t t r i b u t e d to both e x t e r n a l f i l m and i n t e r n a l d i f f u s i o n a l r e s i s t a n c e s . Particle c o m p o s i t i o n , s i z e , shape and p o r e v o l u m e a r e i m p o r t a n t f a c t o r s in d e t e r m i n i n g the e f f e c t i v e n e s s of these k i n d s of s u p p o r t s f o r e n z y m e s (38, 39). G r e e n f i e l d and L a u r e n c e (39) and B o u i n et a l . (40) point out that g l u c o s e o x i d a s e i m m o b i l i z e d s i m u l t a n e o u s l y with c a t a l a s e s i g n i f i c a n t l y extends the u s e f u l n e s s of the g l u c o s e o x i d a s e a c t i v i t y by d e c o m p o s i n g the h y d r o g e n p e r o x i d e f o r m e d in the g l u c o s e o x i d a s e r e a c t i o n . I m m o b i l i z e d c a t a l a s e and g l u c o s e o x i d a s e u n d e r g o s u b s t r a t e and p r o d u c t i n a c t i v a t i o n , r e s p e c t i v e l y , b y h y d r o g e n p e r o x i d e (41). A s a c o n s e q u e n c e the i n d u s t r i a l p e r f o r m a n c e of these e n z y m e s w i l l p r o b a b l y be d e t e r m i n e d by h o w w e l l this i n a c t i v a t i o n c a n be r e d u c e d and controlled. Fibrous Proteins. T h e f i b r o u s p r o t e i n s offer a n u m b e r of p o t e n t i a l advantages as e n z y m e s u p p o r t s for food a p p l i c a t i o n s . T h e y a r e n a t u r a l m a t e r i a l s , with good m e c h a n i c a l p r o p e r t i e s , offer a l o w r e s i s t a n c e to s u b s t r a t e d i f f u s i o n , and h a v e a n open i n t e r n a l s t r u c t u r e with m a n y p o t e n t i a l b i n d i n g s i t e s f o r e n z y m e a t t a c h m e n t and s t a b i l i z a t i o n . T h e y a r e abundant and i n e x p e n s i v e and can be p r o c e s s e d into f i l m s , m e m b r a n e s and other f o r m s that r e t a i n t h e i r s t r u c t u r a l i d e n t i t y . O f the f i b r o u s p r o t e i n s the c o l l a g e n s h a v e b e e n the m o s t w i d e l y studied as e n z y m e s u p p o r t s (42, 43). Collagen proteins in c o m b i n a t i o n w i t h e l a s t i n , m u c o p o l y s a c c h a r i d e s and m i n e r a l s a l t s f o r m the c o n n e c t i v e t i s s u e r e s p o n s i b l e f o r the s t r u c t u r a l i n t e g r i t y of the a n i m a l b o d y . T h e d i s t i n c t i v e c h a r a c t e r i s t i c of the a m i n o a c i d c o m p o s i t i o n of a v a r i e t y of v e r t e b r a t e c o l l a g e n s i s a h i g h g l y c i n e content w h i c h i s c l o s e to o n e - t h i r d of the total n u m b e r of r e s i d u e s . C o l l a g e n s a r e a l s o r i c h i n the i m i n o a c i d s p r o l i n e and h y d r o x y p r o l i n e w h i c h c a n a m o u n t to 20-25% of the total n u m b e r of r e s i d u e s . T h e unique t r i p l e s t r a n d e d h e l i c a l s t r u c t u r e of c o l l a g e n is c l o s e l y r e l a t e d to the h i g h content of g l y c i n e and i m i n o a c i d r e s i d u e s . T h e r e i s a c o m p l e x r e g u l a r i t y to the o r g a n i z a t i o n of the c o l l a g e n m o l e c u l e s into m i c r o f i b r i l s that r e s u l t s i n the p r e s e n c e of h o l e s s p a c e d at r e p e a t i n g i n t e r v a l s a l o n g the l e n g t h of the m i c r o f i b r i l . It i s b e l i e v e d that these r e g i o n s a r e the p r i m a r y s i t e s f o r e n z y m e b i n d i n g (44). C o l l a g e n s w e l l s i n the p r e s e n c e of



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w a t e r , i s h y d r o p h i l i c , and at n e u t r a l p H takes up m o r e than i t s own weight of w a t e r . I m m o b i l i z a t i o n of e n z y m e s on c o l l a g e n i n v o l v e s the f o r m a t i o n of a n e t w o r k of n o n - c o v a l e n t bonds s u c h a s s a l t l i n k a g e s ( i o n i c i n t e r a c t i o n s ) , h y d r o g e n bonds and V a n d e r W a a l s i n t e r a c t i o n s a c t i n g together between c o l l a g e n and the e n z y m e (43). W h i l e i n d i v i d u a l l y these bonds a r e weak, taken together they f o r m a v e r y stable n e t w o r k between e n z y m e and collagen. C o l l a g e n - e n z y m e c o m p l e x e s h a v e b e e n p r e p a r e d by t h r e e d i f f e r e n t p r o c e d u r e s : (1) i m p r e g n a t i n g a p r e s w o l l e n m e m b r a n e w i t h e n z y m e , (2) m a c r o m o l e c u l a r c o m p l e x a t i o n i n w h i c h a n e n z y m e - c o l l a g e n d i s p e r s i o n at the d e s i r e d p H i s c a s t and d r i e d and then r e h y d r a t e d for u s e , and (3) e l e c t r o c o d e p o s i t i o n i n w h i c h e n z y m e i s d e p o s i t e d i n a c o l l a g e n m e m b r a n e u n d e r the i n f l u e n c e of an e l e c t r i c f i e l d . The complexes can subsequently be t r e a t e d w i t h g l u t a r a l d e h y d e to i n d u c e c r o s s l i n k s to s t r e n g t h en the m e m b r a n e and the b o n d i n g of the e n z y m e to the c o l l a g e n . C o l l a g e n - e n z y m e m e m b r a n e s have b e e n c o i l e d and p l a c e d i n a c y l i n d e r with s u i t a b l e s p a c e r s to m a k e efficient continuous r e a c t o r s f o r e n z y m e c o n v e r s i o n . A l a r g e n u m b e r of e n z y m e s , i n c l u d i n g whole c e l l s , h a v e b e e n i m m o b i l i z e d on c o l l a g e n m e m branes. T h i s technique s e e m s a p p l i c a b l e i n g e n e r a l to e n z y m e immobilization. K e r a t i n i s a n o t h e r f i b r o u s p r o t e i n that h a s b e e n e x a m i n e d a s an e n z y m e s u p p o r t . K e r a t i n i s found i n the outer l a y e r s of v e r t e b r a t e s , is un r e a c t i v e t o w a r d the e n v i r o n m e n t and i s m e c h a n i c a l l y s t r o n g and d u r a b l e . L i k e the c o l l a g e n s , k e r a t i n s v a r y d e p e n d i n g on t h e i r s o u r c e . T h e y h a v e a h i g h content of the s u l f u r c o n t a i n i n g a m i n o a c i d s and c r o s s l i n k i n g p r o v i d e d b y the S-S bond of c y s t i n e w h i c h m a i n t a i n s s t r u c t u r a l i n t e g r i t y . Stanley and c o - w o r k e r s (45) h a v e r e c e n t l y p r e p a r e d a g r a n u l a r k e r a t i n f r o m f e a t h e r m e a l , a b y p r o d u c t of the p o u l t r y i n d u s t r y , and h a v e s u c c e s s f u l l y bound l a c t a s e to the g r a n u l e s w i t h g l u t a r a l d e h y d e . F e a t h e r k e r a t i n i n r e d u c e d f o r m h a s s o m e unique a t t r i butes w o r t h e x p l o i t i n g . T h e h i g h c o n c e n t r a t i o n of f r e e s u l f h y d r y l g r o u p s c o u l d be u s e d i n p u r i f y i n g s u l f u r - c o n t a i n i n g e n z y m e s t h r o u g h the technique of t h i o l - d i s u l f i d e i n t e r c h a n g e (46) o r s i m p l y by a d s o r p t i o n i n s o m e c a s e s . U r e a s e h a s b e e n i m m o b i l i z e d on f e a t h e r k e r a t i n b y a d s o r p t i o n (45). P h e n o l i c R e s i n s . A r e s i n made f r o m phenol and f o r m a l d e hyde h a s b e e n found to be a n e x c e l l e n t s u p p o r t f o r s o m e e n z y m e s by s e v e r a l i n v e s t i g a t o r s (47, 48, 49). A r e s i n of this type i s c o m m e r c i a l l y a v a i l a b l e f r o m D i a m o n d S h a m r o c k C h e m i cal C o m p a n y , Redwood C i t y , C a l i f o r n i a as Duolite E S - 7 6 2 . T h i s p o r o u s r e s i n i s r e l a t i v e l y stable to a c i d s , b a s e s a n d m o s t organic solvents. T h e 10-50 m e s h p a r t i c l e s a r e r i g i d , i r r e g u l a r and g r a n u l a r , and r e s i s t a n t attrition. They have a pore v o l u m e of a b o u £ 0. 6^cm p e r c m of r e s i n and a s u r f a c e a r e a of about 100 m / c m . T h i s type of r e s i n i s u s e d a s a n a d s o r b a n t



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f o r p r o t e i n s , a r o m a t i c s and other c o m p o u n d s a n d a s s u c h it w i l l b i n d e n z y m e s without f u r t h e r t r e a t m e n t . H o w e v e r , the a d d i t i o n of g l u t a r a l d e h y d e to the r e s i n e i t h e r b e f o r e o r a f t e r a p p l i c a t i o n of the e n z y m e c a n i n c r e a s e the r e t e n t i o n of e n z y m e a c t i v i t y s i g nificantly* I m m o b i l i z a t i o n is r a p i d at r o o m t e m p e r a t u r e and can be done i n a b a t c h p r o c e s s i n a n open v e s s e l o r with the r e s i n p a c k e d i n a c o l u m n . In m o s t c a s e s the e n z y m e can be r e m o v e d f r o m the r e s i n b y t r e a t m e n t w i t h d i l u t e a c i d a n d / o r b a s e and new e n z y m e a d s o r b e d to the s u p p o r t . T h i s r e s i n has been s u c c e s s f u l l y u s e d to i m m o b i l i z e l a c t a s e (47, 48), α-amylase (49), t r y p s i n (50) and other e n z y m e s . M o d i f i c a t i o n s of the p h e n o l f o r m a l d e h y d e r e s i n a r e a l s o p o s s i b l e . R e s i n s substituted with v a r i o u s p r i m a r y , s e c o n d a r y and t e r t i a r y m e t h y l a m i n o groups a r e a l s o available. T h e s e a n i o n exchange r e s i n s h a v e b e e n u s e d to i m m o b i l i z e i n v e r t a s e (48), g l u c o s e i s o m e r a s e and a s p a r t a s e (51). T h e a d s o r p t i o n p r o c e s s with the u n s u b s t i t u t e d r e s i n m o s t p r o b a b l y i n v o l v e s the p h e n o l i c r i n g s i n t e r a c t i n g w i t h a r o m a t i c a m i n o a c i d s i n the p r o t e i n . T h i s type of i n t e r a c t i o n of p r o t e i n s with p h e n o l i c c o m p o u n d s i s a l s o o b s e r v e d i n t h e i r r e a c t i o n w i t h tannins and i n p a r t i c u l a r tannic a c i d to f o r m i n s o l u b l e c o m p l e x e s . S u c h c o m p l e x e s w i t h i n v e r t a s e and l a c t a s e a r e e n z y m a t i c a l l y a c t i v e a n d r e m a i n i n s o l u b l e i f the c o m p l e x i s s t a b i l i z e d with g l u t a r a l d e h y d e (48). It is a l s o n e c e s s a r y to d e p o s i t this c o m p l e x on a s u p p o r t s u c h a s C e l i t e f o r c o l u m n o p e r a t i o n . W h i l e these l i m i t a t i o n s on the u s e of tannins to p r e c i p i tate e n z y m e s a p p e a r s g r e a t , the c o m b i n a t i o n s of different t a n n i n s and s u p p o r t s that c o u l d be t r i e d s u g g e s t s that for s o m e a p p l i c a t i o n s this a p p r o a c h c o u l d be u s e f u l . H y d r o g e l s . T h e m e t h o d of g e l e n t r a p m e n t i n v o l v e s the f o r m a t i o n of a c r o s s l i n k e d p o l y m e r n e t w o r k i n the p r e s e n c e of an e n z y m e o r m i c r o b i a l c e l l . T h i s technique s e e m s to be p a r t i c u l a r l y a t t r a c t i v e f o r the i m m o b i l i z a t i o n of whole c e l l s . With whole c e l l i m m o b i l i z a t i o n , the c o s t l y e n z y m e p u r i f i c a t i o n p r o c e s s e s c a n be a v o i d e d . S e q u e n t i a l r e a c t i o n s c a n be c a r r i e d out u t i l i z i n g the v a r i o u s e n z y m e s and c o f a c t o r s w h i c h a r e left intact i n the m i c r o b i a l c e l l s . W i t h i n the m i c r o b i a l c e l l s m a n y e n z y m e s a r e a l r e a d y i m m o b i l i z e d and c o m p a r t m e n t a l i z e d i n o r d e r to c a r r y out c o m p l e x m e t a b o l i c and synthetic r e a c t i o n s . By entrapping cells in a hydrophilic p o l y m e r network, a sup p o r t i n g s t r u c t u r e can be p r o v i d e d that h a s l i t t l e effect on c e l l properties. T h e p o l y m e r m o s t often u s e d f o r e n t r a p m e n t i s p o l y a c r y l amide. T h e c r o s s l i n k i n g agent u s e d to f o r m a t h r e e d i m e n s i o n al network is usually Ν , N - m e t h y l e n e b i s ( a c r y l a m i d e ) . Poly m e r i z a t i o n i s c a r r i e d out i n a n aqueous s o l u t i o n c o n t a i n i n g e n z y m e o r m i c r o b i a l c e l l , m o n o m e r , c r o s s l i n k i n g agent, and free r a d i c a l initiator. A three d i m e n s i o n a l p o l y m e r network is f o r m e d entrapping enzyme or m i c r o b i a l c e l l . T a n a b e S e i y a k u C o m p a n y i n J a p a n h a s d e v e l o p e d two 1



7.

OLSON

AND

KORUS

Immobilized Enzymes

109

p r o c e s s e s f o r a m i n o a c i d p r o d u c t i o n b a s e d on m i c r o b i a l c e l l e n t r a p m e n t i n p o l y a c r y l a m i d e g e l s . In one p r o c e s s , L - a s p a r t i c a c i d i s p r o d u c e d f r o m f u m a r i c a c i d u s i n g the a s p a r t a s e a c t i v i t y of e n t r a p p e d E . c o l i b a c t e r i a l c e l l s . In the other p r o c e s s , L m a l i c a c i d i s p r o d u c e d f r o m f u m a r i c a c i d u s i n g the f u m a r a s e a c t i v i t y of B r e v i b a c t e r i u m a m m o n i a gene s (52). Β . a m m o n i a genes c e l l s e n t r a p p e d i n p o l y a c r y l a m i d e gels h a v e a l s o b e e n studied f o r the s y n t h e s i s of c o e n z y m e A f r o m pantothenic a c i d , c y s t e i n e , and A T P . This synthesis uses five sequential enzymic steps and i l l u s t r a t e s the advantage of u s i n g i m m o b i l i z e d whole c e l l s f o r m u l t i - s t e p s y s t e m s (53). A l t h o u g h i t i s u s u a l l y a s s u m e d that e n z y m e s e n t r a p p e d i n p o l y a c r y l a m i d e gels r e m a i n unattached to the g e l m a t r i x and r e t a i n t h e i r n a t i v e c o n f o r m a t i o n , this m a y not a l w a y s be t r u e . H a r r i s o n has shown that the e n z y m e g l u c o s e 6-phosphate dehy d r o g e n a s e i s bound c o v a l e n t l y to the g e l m a t r i x v i a f r e e s u l f h y d r y l o r a m i n o g r o u p s d u r i n g gel e n t r a p m e n t (54). E n z y m e i m m o b i l i z a t i o n by g e l e n t r a p m e n t h a s b e e n r e c e n t l y r e v i e w e d (55). A l t h o u g h m o s t e n z y m e e n t r a p m e n t has been i n p o l y a c r y l a m i d e , o t h e r h y d r o g e l s m a y be s u p e r i o r with r e g a r d to m e c h a n i c a l s t r e n g t h and e l i m i n a t i o n of e n z y m e l e a k a g e . A l a r g e n u m b e r of e n z y m e s h a v e b e e n e n t r a p p e d i n poly( 2 - h y d r o x y e t h y l m e t h y l a c r y l a t e ) g e l s , (5_6). T h i s g e l m a y be p r e f e r r e d f o r s o m e a p p l i c a t i o n s b e c a u s e of i t s s u p e r i o r m e c h a n i c a l s t r e n g t h . E n z y m e s h a v e a l s o b e e n i m m o b i l i z e d u s i n g a l a r g e n u m b e r of synthetic m o n o m e r s and p o l y m e r s s u c h a s Ν-vinyl p y r r o l i d one, p o l y ( v i n y l a l c o h o l ) , and p o l y ( v i n y l p y r r o l i d o n e ) u n d e r r a d i a n t r a y i r r a d i a t i o n (57). Whole y e a s t c e l l s h a v e b e e n e n t r a p p e d i n s p h e r i c a l a g a r p e l l e t s and u s e d f o r s u c r o s e h y d r o l y s i s (58). H o l l o w F i b e r s . A n i m m o b i l i z a t i o n technique w h i c h o f f e r s s e v e r a l a d v a n t a g e s f o r food s y s t e m s i s the u s e of h o l l o w f i b e r s . Two types of h o l l o w f i b e r d e v i c e s that can be u s e d f o r e n z y m e i m m o b i l i z a t i o n a r e the i s o t r o p i c , c e l l u l o s i c f i b e r s m a d e by Dow C h e m i c a l C o m p a n y and the a n i s o t r o p i c , n o n c e l l u l o s i c f i b e r s m a d e by A m i c o n C o r p o r a t i o n and R o m i c o n C o r p o r a t i o n . The Dow s e m i p e r m e a b l e h o l l o w f i b e r s a r e m a d e of c e l l u l o s e a c e t a t e o r other c e l l u l o s i c m a t e r i a l s . T h e y a r e h o m o g e n e o u s ( i s o t r o p i c ) and p e r m i t p e r m e a t i o n of s o l u t e o r s o l v e n t f r o m e i t h e r s i d e of the f i b e r . A m i c o n and R o m i c o n f i b e r s a r e a n i s o t r o p i c and c o m p o s e d of an a p p r o x i m a t e l y 0. 5 μιη t h i c k s e m i p e r m e a b l e m e m b r a n e s u p p o r t e d by a t h i c k o p e n - c e l l e d sponge ( F i g u r e 2). T h e X50 h o l l o w f i b e r s a r e c o p o l y m e r s of p o l y ( v i n y l c h l o r i d e ) and p o l y a c r y l o n i t r i l e and h a v e a n o m i n a l m o l e c u l a r weight c u t - o f f of 50,000. The P10 f i b e r s a r e p o l y s u l f o n e p o l y m e r s with a n o m i n a l m o l e c u l a r weight c u t - o f f of 10, 000. T h e i m m o b i l i z a t i o n technique often u s e d with the A m i c o n and R o m i c o n h o l l o w f i b e r s c o n s i s t s of p h y s i c a l l y e n t r a i n i n g an aque ous p r e p a r a t i o n of e n z y m e or whole c e l l s w i t h i n the p o r o u s sponge r e g i o n of the f i b e r s . S u b s t r a t e s o l u t i o n i s then p a s s e d


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AXIAL CROSS SECTION


Figure 2.

RADIAL CROSS SECTION

Structure of assymmetric hollow fiber

t h r o u g h the f i b e r l u m e n g i v i n g a continuous flow e n z y m e r e a c tor. T h e e n z y m e - s u b s t r a t e s y s t e m m u s t be s e l e c t e d so that the e n z y m e i s too l a r g e to p a s s t h r o u g h the f i b e r m e m b r a n e w h i l e the s u b s t r a t e i s s u f f i c i e n t l y s m a l l so a s to p a s s t h r o u g h the membrane. A theoretical model describing enzymic catalysis using hollow fiber m e m b r a n e s has been developed by Waterland et a l . (59). R e a c t o r c o n v e r s i o n data f o r β - gala c to s i d a se a g r e e w e l l w i t h p r e d i c t i o n s obtained f r o m the t h e o r e t i c a l m o d e l (60). H o l l o w fiber enzyme r e a c t o r s have also been used for i m m o b i l i z a t i o n of g l u c o s e i s o m e r a s e (61), cz-galactosidase a n d i n v e r t a s e (62). T h e s t a b i l i t y of e n z y m e s i m m o b i l i z e d i n h o l l o w f i b e r r e a c t o r s i s a p p r o x i m a t e l y the s a m e as f o r the c o r r e s p o n d i n g e n z y m e s i n f r e e s o l u t i o n . H o w e v e r , the R o m i c o n and A m i c o n P 1 0 h o l l o w f i b e r s r e q u i r e a p r e c o n d i t i o n i n g with a n i n e r t p r o t e i n b e f o r e u s e i n o r d e r to a v o i d e n z y m e i n a c t i v a t i o n on contact w i t h the f i b e r s (61, 62). S o m e of the advantages of u s i n g h o l l o w f i b e r s f o r e n z y m e i m m o b i l i z a t i o n a r e : h i g h throughput with a l o w p r e s s u r e d r o p can be a c h i e v e d , n o e n z y m e m o d i f i c a t i o n i s r e q u i r e d and t h e r e i s n o p r o d u c t c o n t a m i n a t i o n f r o m c h e m i c a l s u s e d i n the i m m o b i l i z a t i o n p r o c e s s , h o l l o w f i b e r c a r t r i d g e s c a n be e a s i l y c l e a n e d and r e l o a d e d w i t h e n z y m e , a n d the f i b e r s have a l a r g e s u r f a c e t o - v o l u m e r a t i o a n d a thin m e m b r a n e w h i c h offers a l o w r e s i s t a n c e to s u b s t r a t e t r a n s p o r t . Microbial Pellets. In a s u b m e r g e d c u l t u r e of f i l a m e n t o u s m i c r o o r g a n i s m s , the m y c e l i a a r e l i k e l y to a g g r e g a t e to f o r m pellets. M e d i a c o m p o s i t i o n and p H of the f e r m e n t a t i o n m e d i a can d e t e r m i n e whether pulpy m y c e l i a o r pellets a r e f o r m e d (63). M i c r o b i a l p e l l e t s c a n be v i e w e d a s i m m o b i l i z e d whole c e l l s w h e r e the i n t r a c e l l u l a r e n z y m e s a r e a v a i l a b l e f o r u t i l i z a tion. T h e s e u n t r e a t e d c e l l s c a n be d i r e c t l y u s e d a s b i o c a t a l y s t s



7.

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A N D KORUS

Immobilized Enzymes

111

for i n d u s t r i a l p r o c e s s e s . T h i s e l i m i n a t e s the n e e d f o r e n z y m e p u r i f i c a t i o n and g r e a t l y s i m p l i f i e s the i m m o b i l i z a t i o n p r o c e s s . P e l l e t s p r o d u c e d by f e r m e n t a t i o n of the fungus M o r t i e r e l l a v i n a c e a e have b e e n s u c c e s s f u l l y u s e d i n the beet s u g a r i n d u s t r y a s a n e n z y m e s o u r c e of ar-galactosidase. This enzyme hydrol y z e s the i n d i g e s t i b l e s u g a r r a f f i n o s e to s u c r o s e . Raffinose r e d u c e s s u g a r y i e l d by r e t a r d i n g the r a t e of s u c r o s e p r e c i p i t a tion f r o m beet s u g a r m o l a s s e s . K i n e t i c studies of or-galactosidase c o n t a i n i n g p e l l e t s of M . v i n a c e a e show that the b e h a v i o r of p e l l e t s is s i m i l a r to that of gel e n t r a p p e d e n z y m e s (64). T h e r e is a decrease in enzyme a c t i v i t y w i t h i n c r e a s i n g p e l l e t s i z e above 0. 25 m m b e c a u s e of d i f f u s i o n a l effects. F o r p e l l e t s s m a l l e r than about 0. 25 m m i n d i a m e t e r , d i f f u s i o n a l effects a r e a b s e n t . The effectiveness f a c t o r s found e x p e r i m e n t a l l y f o r p e l l e t s of M . v i n a c e a e with ag a l a c t o s i d a s e a c t i v i t y w e r e found to a g r e e f a i r l y w e l l w i t h a theoretical m o d e l d e s c r i b i n g intraparticle diffusion in s p h e r i c a l particles. C o m m e r c i a l A p p l i c a t i o n of I m m o b i l i z e d E n z y m e s D e t a i l e d i n f o r m a t i o n on c o m m e r c i a l p r o c e s s e s u s i n g i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g i s g e n e r a l l y p r o p r i e t a r y . F o r s o m e p r o c e s s e s the only a v a i l a b l e i n f o r m a t i o n i s the m i c r o o r g a n i s m f r o m w h i c h the e n z y m e u s e d was obtained and a g e n e r a l d e s c r i p t i o n of the i m m o b i l i z a t i o n m e t h o d . T h e c o m m e r c i a l a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s p r e s e n t e d i n this s e c t i o n a r e w e l l d o c u m e n t e d and r e p r e s e n t s i g n i f i c a n t a d v a n c e s in p r o c e s s d e v e l o p m e n t i n the food i n d u s t r y . Aminoacylase. T h e f i r s t l a r g e s c a l e use of i m m o b i l i z e d e n z y m e s was the T a n a b e S e i y a k u C o m p a n y p r o c e s s f o r L - a m i n o a c i d production in Japan. This p r o c e s s uses aminoacylase a d s o r b e d on D E A E - S e p h a d e x f o r the c o n t i n u o u s , a u t o m a t i c a l l y c o n t r o l l e d p r e p a r a t i o n of L - m e t h i o n i n e , L - p h e n y l a l a n i n e , L tryptophane and L - v a l i n e f r o m r a c e m i c m i x t u r e s of those a m i n o acids. T h e r e d u c e d c o s t s of e n z y m e , s u b s t r a t e and l a b o r h a v e cut p r o d u c t i o n c o s t s of the i m m o b i l i z e d e n z y m e p r o c e s s to 60% of that f o r the c o n v e n t i o n a l b a t c h p r o c e s s u s i n g s o l u b l e e n z y m e s (65, 66). A p r o c e s s flow sheet f o r the continuous p r o d u c t i o n of L a m i n o a c i d f r o m r a c e m i c a c e t y l - D , L - a m i n o a c i d i s shown i n F i g u r e 3. T h e a c y l a t e d a m i n o a c i d m i x t u r e i s fed to a c o l u m n c o n t a i n i n g a m i n o a c y l a s e i m m o b i l i z e d b y a d s o r p t i o n onto D E A E Sephadex. T h i s e n z y m e h y d r o l y z e s only the a c e t y l - L - a m i n o acid. T h e r e s u l t i n g m i x t u r e of a c e t y l - D - a m i n o a c i d and L a m i n o a c i d c a n be s e p a r a t e d b y c r y s t a l l i z a t i o n b e c a u s e of solubility differences. L - m e t h i o n i n e h a s been p r o d u c e d w i t h a r e p o r t e d output of 20 m e t r i c tons p e r m o n t h (67).


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ACETYLDL-AMINOACID

CRYSTALLIZER

RACEMIZATION TANK

EVAPORATOR IMMOBILIZED


HEAT EXCHANGER

AMINOACYLASE COLUMN

CRYSTALLINE L-AMINO

ACID

Figure 3.Process flow sheet for the continuous production of \.-amino acid from racemic acetyl-O-^amino acid

Glucose Isomerase. U n t i l 1938 c o m m e r c i a l c o r n s y r u p s w e r e p r o d u c e d by a c i d h y d r o l y s i s of c o r n s t a r c h . This process p r o d u c e d a s y r u p with a d e x t r o s e - e q u i v a l e n t ( D . E . ) of 5 6 - 5 8 . A s w e e t e r s y r u p was not p r o d u c e d b e c a u s e h i g h e r c o n v e r s i o n s by a c i d h y d r o l y s i s gave b i t t e r f l a v o r s . W i t h the i n t r o d u c t i o n of s a c c h a r i f y i n g e n z y m e s it b e c a m e p o s s i b l e to i n c r e a s e the D . E . without p r o d u c i n g b i t t e r f l a v o r s . The glucoamylase currently u s e d f o r s t a r c h s a c c h a r i f i c a t i o n p r o d u c e c o r n s y r u p s with a D . E . of 9 5 - 9 7 . T h i s h i g h d e x t r o s e c o r n s y r u p , w h i c h i s 70-75% as sweet as s u c r o s e , c a n be t r a n s f o r m e d to a p r o d u c t w h i c h i s e q u i v a l e n t i n s w e e t n e s s to s u c r o s e by a p a r t i a l i s o m e r i z a t i o n of g l u c o s e ( d e x t r o s e ) to f r u c t o s e . T h i s i s o m e r i z a t i o n has b e e n c a r r i e d out on a c o m m e r c i a l s c a l e s i n c e 1968. The c o m m e r c i a l a p p l i c a t i o n of i m m o b i l i z e d g l u c o s e i s o m e r a s e to p r o d u c e h i g h f r u c t o s e c o r n s y r u p i s c u r r e n t l y the l a r g e s t v o l u m e u s e of a n i m m o b i l i z e d e n z y m e i n the w o r l d . A p p r o x i m a t e l y one b i l l i o n pounds of h i g h f r u c t o s e s y r u p w e r e p r o d u c e d i n 1974 (9). The c o m m e r c i a l p r o c e s s for high fructose c o r n s y r u p p r o d u c t i o n i s shown i n F i g u r e 4. The process involves liquefying r a w c o r n s t a r c h , s a c c h a r i f y i n g the s t a r c h to a h i g h d e x t r o s e s y r u p u s i n g a c i d a n d e n z y m i c h y d r o l y s i s , and r e f i n i n g the syrup. T h e c a r b o n and i o n exchange r e f i n e d g l u c o s e s y r u p c o n tains about 93% D - g l u c o s e on a d r y b a s i s . T h e r e m a i n i n g 7% i s m a d e up of o t h e r c o r n s y r u p s a c c h a r i d e s . T h i s glucose syrup i s p r e p a r e d for i s o m e r i z a t i o n by the a d d i t i o n of r e q u i r e d s a l t s (Co and Mg) and a d j u s t m e n t of p H . T h e g l u c o s e s y r u p i s p u m p e d t h r o u g h a s e r i e s of s h a l l o w bed r e a c t o r s w h i c h c o n t a i n g l u c o s e i s o m e r a s e a d s o r b e d onto D E A E - c e l l u l o s e . T h e flow r a t e i s c o n t r o l l e d so that the p r o d u c t c o n t a i n s about 42% f r u c tose. T h e flow r a t e c a n be r e d u c e d as i s o m e r a s e a c t i v i t y


7.

OLSON

A N D

KORUS

113

Immobilized Enzymes

LIGHT EVAPORATION

HEAVY EVAPORATION

SALTS pH

COOLING & STORAGE

ADJUSTMENT


CARBON REFINING

Figure 4.

CD

High-fructose corn syrup process flow sheet

d i m i n i s h e s i n o r d e r to m a i n t a i n this c o n v e r s i o n . T h e f r u c t o s e s y r u p i s then r e f i n e d and c o n c e n t r a t e d by e v a p o r a t i o n . T h e r e fined h i g h f r u c t o s e c o r n s y r u p i s s u b s t a n t i a l l y c o l o r l e s s , a s h f r e e and h a s l i t t l e tendency to f o r m c o l o r on s t o r a g e (68). A h i g h e r f r u c t o s e content can be obtained by s e p a r a t i n g g l u c o s e and f r u c t o s e i n i o n exchange c o l u m n s , t h e r e b y p r o d u c i n g 90% f r u c t o s e s y r u p s on a l a r g e s c a l e w i t h h i g h y i e l d s (69). These s y r u p s a r e now c o m m e r c i a l l y a v a i l a b l e . cy-Galactosidase. T h e beet s u g a r i n d u s t r y u s e s i m m o b i l i z e d a- gala c to s i d a se to h y d r o l y z e the r a f f i n o s e i n s u g a r b e e t s . This e n z y m e c a n be h e l d w i t h i n p e l l e t s of the fungus M . v i n a c e a e which are formed under specific fermentation conditions. D i l u t e d s u g a r beet m o l a s s e s i s p a s s e d t h r o u g h a r e a c t o r c o n t a i n i n g the p e l l e t s . T h e p e l l e t s a r e v e r y c o m p r e s s i b l e and c a n not be r e a d i l y p a c k e d i n a c o l u m n without an e x c e s s i v e p r e s s u r e b u i l d - u p d u r i n g continuous o p e r a t i o n . A n e n z y m e r e a c t o r that has b e e n d e v e l o p e d f o r the c o m m e r c i a l p r o c e s s (70) c o n s i s t s of a h o r i z o n t a l vat d i v i d e d into s e v e r a l c h a m b e r s , e a c h b e i n g a g i tated, a n d e a c h s e p a r a t e d by a s c r e e n to p r e v e n t the p a s s a g e of p e l l e t s f r o m one c h a m b e r to a n o t h e r . T h e m o l a s s e s i s fed into one end of the r e a c t o r and c o n t a c t s the s u s p e n d e d p e l l e t s as it p a s s e s f r o m c h a m b e r to c h a m b e r . T y p i c a l l y , a s u g a r beet p r o c e s s i n g plant m i g h t p r o c e s s 3000 tons of beets a d a y and obtain w a t e r - s o l u b l e e x t r a c t s r e p r e s e n t ing 600 tons of s u c r o s e and as m u c h as 4 o r 5 tons of raffinose (71). A b o u t 80% of the r a f f i n o s e c a n be h y d r o l y z e d as the m o l a s s e s i s p a s s e d t h r o u g h the a g i t a t e d c h a m b e r s . S i n c e r a f f i n o s e i n t e r f e r e s with the c r y s t a l l i z a t i o n of s u c r o s e , s u c r o s e r e c o v e r y i s i n c r e a s e d by r a f f i n o s e h y d r o l y s i s to a g r e a t e r extent than the a m o u n t of r a f f i n o s e h y d r o l y z e d . E x p e r i e n c e i n J a p a n i n d i c a t e d


114

ENZYMES

IN FOOD AND B E V E R A G E

PROCESSING

that p l a n t throughput was i n c r e a s e d by at l e a s t 5 % and r e c o v e r i e s of s u c r o s e i n c r e a s e d (71). T h e r e i s a l s o i n t e r e s t i n or-ga l a c to s i d a se f o r h y d r o l y s i s of the or-galactosides, r a f f i n o s e , s t a c h y o s e and v e r b a s c o s e , that a r e c o n s i d e r e d to be p a r t i a l l y r e s p o n s i b l e f o r the f l a t u l e n c e a s s o c i a t e d w i t h d r y b e a n p r o d u c t s s u c h as s o y b e a n m i l k (62, 72). H o w e v e r , t h e r e h a v e b e e n no r e p o r t s of a c o m m e r c i a l application. R i b o n u c l e a s e . A r e c e n t a p p l i c a t i o n of i m m o b i l i z e d e n z y m e s i n J a p a n i s the p r o d u c t i o n of 5'-mononucleotide f l a v o r e n h a n c e r s (34). I n o s i n e - 5 - m o n o p h o s p h a t e (IMP) and guanosine-5'-mono phosphate ( G M P ) a c t s y n e r g i s t i c a l l y w i t h m o n o s o d i u m g l u t a m a t e to e n h a n c e food f l a v o r s . T h e 5'- r i b o n u c l e o t i d e s , a d e n o s i n e - 5 ' monophosphate (AMP), cytidine-5'-monophosphate ( C M P ) , u r i d i n e - 5 ' - m o n o p h o s p h a t e ( U M P ) and G M P , are produced f r o m r i b o n u c l e i c a c i d s u s i n g the r i b o n u c l e a s e 5 * - p h o s p h o d i e s t e r a s e i m m o b i l i z e d on a p o r o u s c e r a m i c support. I M P _ i s p r o d u c e d b y the r e p l a c e m e n t of the a m i n o g r o u p of A M P with a h y d r o x y l group. T h i s r e a c t i o n i s c a t a l y z e d by 5'-adenylate d e a m i n a s e i m m o b i l i z e d on a p o r o u s c e r a m i c s u p p o r t .


f

RNA—=τ—r r—τ: : 5 -phosphodiesterase

AMP

• 5'-ribonucleotides Μ Ρ , GMP, CMP,

. -ζ : • u 5 -adenylate d e a m i n a s e

Γ

Ί

(

Α

UMP)

IMP

F r o m these o b s e r v a t i o n s on what h a s s u c c e e d e d i n the f i e l d of i m m o b i l i z e d e n z y m e s a p p l i e d to food p r o c e s s i n g we w o u l d suggest that f o r l a r g e v o l u m e , l o w unit p r i c e i t e m s , the m o r e s i m p l e i m m o b i l i z a t i o n s c h e m e s on i n e x p e n s i v e food grade sup p o r t s a r e n e c e s s a r y . A d s o r p t i o n on D E A E c e l l u l o s e and s t a b i l i z e d m i c r o b i a l p e l l e t s h a v e s u c c e e d e d . O f c o u r s e , a s the v a l u e of the p r o d u c t goes up a m o r e c o m p l e x s c h e m e m a y be p o s s i b l e . P o t e n t i a l New

A p p l i c a t i o n s of I m m o b i l i z e d

Enzymes

S o m e e x a m p l e s of the m a n y p r o p o s e d u s e s of i m m o b i l i z e d e n z y m e s to food p r o c e s s i n g a r e l i s t e d i n T a b l e I. E a c h of these a p p l i c a t i o n s i s a s i g n i f i c a n t s u b j e c t i n and of i t s e l f . We h a v e s e l e c t e d a few p r o p o s a l s to d i s c u s s i n m o r e d e t a i l as i l l u s t r a t i v e of what m i g h t be done w i t h i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g i n the f u t u r e . Immobilized Starch H y d r o l y z i n g E n z y m e Systems. Sugar syrups prepared f r o m enzymatically hydrolyzed starch are i m p o r t a n t i n g r e d i e n t s i n m a n y m o d e r n food p r o d u c t s . S y r u p s a r e a v a i l a b l e c o n t a i n i n g a wide range of s u g a r s w i t h v a r y i n g sweet-


7.

OLSON

AND

T a b l e I.


New

KORUS

115

Immobilized Enzymes

P r o p o s e d U s e s of I m m o b i l i z e d Processing

Application

E n z y m e s to F o o d

Examples

Sugar h y d r o l y s i s

F o r fermentation processes l a c t a s e on whey, p r i o r to wine fermentation

Clarification or haze removal

In b e e r and wine i n d u s t r i e s u s e papain, b r o m e l a i n , and o t h e r proteases

Sterilization

L y s o z y m e to r e m o v e m i c o o r g a n i s m s from air C a t a l a s e to r e m o v e e x c e s s H^O^

Food stabilization

Oxygen r e m o v a l with glucose oxidasecatalase system

Prevent gelling

In f r o z e n o r a n g e j u i c e c o n c e n t r a t e s use pectic enzymes

Flavor modification

D e b i t t e r i n g p r o t e i n s with p r o t e a s e s Naringinase for grapefruit juice Limonate dehydrogenase f o r citrus juices

Nutritional improvement

P l a s t e i n r e a c t i o n to add a m i n o a c i d to protein Cellulases, hemicellulases, pectina s e s , etc, , to i m p r o v e food d i g e s t i bility

Removing undesirable factors

Decaffeinating coffee R e m o v i n g tannins f r o m tea

n e s s and f u n c t i o n a l p r o p e r t i e s . T h e p r o c e s s e s i n v o l v e d i n p r e p a r i n g these s y r u p s constitute one of the l a r g e s t c o m m e r c i a l u s e s of e n z y m e s . C h a n g e s and i m p r o v e m e n t s i n e n z y m e s o u r c e s and m e a n s of u t i l i z a t i o n c a n thus be of c o n s i d e r a b l e i m p o r t a n c e to the i n d u s t r i e s i n v o l v e d , the q u a l i t y and p r o p e r t i e s of the p r o d u c t s p r o d u c e d and the c o n s u m e r . P o t e n t i a l a d v a n tages f o r u s i n g i m m o b i l i z e d e n z y m e s i n these p r o c e s s e s i n c l u d e l o w e r e n z y m e c o s t s , continuous p r o c e s s e s , l o w e r l a b o r c o s t s , c l e a n e r p r o d u c t s , b e t t e r q u a l i t y c o n t r o l and l e s s a d d i t i o n a l p r o c e s s i n g . W i t h these p o t e n t i a l advantages i t i s not s u r p r i s i n g that w e l l o v e r 100 p a p e r s h a v e b e e n p u b l i s h e d i n the l a s t few y e a r s d e a l i n g w i t h the i m m o b i l i z a t i o n of one o r m o r e of the


116

E N Z Y M E S I N FOOD A N D B E V E R A G E

PROCESSING

e n z y m e s i n v o l v e d i n s t a r c h h y d r o l y s i s . S o m e of this w o r k h a s b e e n r e v i e w e d b y Z a b o r s k y (4) a n d O l s o n and R i c h a r d s o n ( 11). T h e p r o c e s s of p r e p a r i n g s u g a r s y r u p s f r o m s t a r c h c a n be d i v i d e d into s e v e r a l steps ( F i g u r e 5). T h e g e l a t i n i z a t i o n a n d Starch Gelatinization pH 2 Swollen Starch L i q u e f a c t i o n a-Amylase,

p H 6. 5,

85°C


Thinned Starch Sac cha r if i c a ti on G l u c oa m y l a s e p H 3. 5-6, 5, 3 5 - 6 5

Enzymes in Food and Beverage Processing

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