Foundations of Biochemical Engineering - American Chemical Society

21 Molecular Size Distribution of Starch During Enzymatic Hydrolysis 1

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J. E. ROLLINGS , M. R. OKOS, and G. T. TSAO Purdue University, West Lafayette, IN 47907

An appropriate means of determining the mole c u l a r s i z e and molecular s i z e d i s t r i b u t i o n of starch during h y d r o l y s i s i s v i a aqueous s i z e e x c l u s i o n chromatography. This method employs a strong a l k a l i n e mobile phase and porous s t a t i o n a r y gel supports which are s t a b l e to the basic s o l u t i o n s . The separation range of molecular s i z e s i s extended to span from 2 x 10 to 5 x 10 hydrodynamic volume (dl/mole) by coupling s i z e e x c l u s i o n chromatographic columns i n a manner that will maintain both l i n e a r i t y i n molecular s i z e separation with e l u t i o n volume and high separation e f f i c i e n c y . This method i s used to e s t a b l i s h that molecular s i z e d i s t r i b u t i o n , r e a c t i o n extent and r e a c t i o n rate s t r o n g l y depend upon the c r y s t a l l i n e state of the substrate and the a c t i v i t y of the enzyme. The s u s c e p t i b i l i t y of starch to h y d r o l y s i s by α-amylase i s c l o s e l y r e l a t e d to the c r y s t a l l i n e states of the substrate. Starch c r y s t a l s are present both i n the n a t u r a l granule s t r u c t u r e and i n retrograded starch. Due to thermal d e a c t i v a t i o n , the highest o v e r a l l conversion achieved f o r a given amount of enzyme i s obtained at temperatures below the upper l i m i t s of the g e l a t i n i z a t i o n range of s t a r c h . The initial rate of the r e a c t i o n i s greatest at temper atures above the g e l a t i n i z a t i o n range. From these r e s u l t s , a p l a u s i b l e p h y s i c a l model of starch s t r u c t u r e during h y d r o l y s i s i s presented. The model states that various c r y s t a l l i n e states of the constituent starch molecules are present through out the course of degradation. These c r y s t a l l i n e states are associated with the n a t u r a l s t a t e of the 7

1

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Current address: Worcester Polytechnic Institute, Worcester, MA 01609 0097-6156/83/0207-0443$06.00/0 © 1983 American Chemical Society

Blanch et al.; Foundations of Biochemical Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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starch granule as w e l l as with r e c r y s t a l l i z e d states caused by r e t r o g r a d a t i o n . In order to properly describe enzymatic s t a r c h l i q u e f a c t i o n , these c r y s t a l l i n e states must be accounted f o r .

R e c e n t l y , much a t t e n t i o n has been g i v e n t o t h e p r o d u c t i o n o f l i q u i d s w e e t e n e r s as an a l t e r n a t i v e t o cane s u g a r u s i n g i n e x p e n s i v e s t a r c h - c o n t a i n i n g n a t u r a l m a t e r i a l s as t h e p r i m a r y f e e d s t o c k . This s i t u a t i o n e x i s t s i n t h e U n i t e d S t a t e s as t h i s c o u n t r y i s not s e l f s u f f i c i e n t i n the p r o d u c t i o n o f c a n e , b u t must r e l y h e a v i l y on i m p o r t a t i o n m a i n l y f r o m S o u t h A m e r i c a and the C a r i b b e a n . The m a i n s o u r c e o f s t a r c h i n t h e U n i t e d S t a t e s comes f r o m c o r n ( Z e a mays) and t h e l i q u i d s w e e t e n e r c o m m e r c i a l l y produced from t h i s m a t e r i a l i s c a l l e d h i g h f r u c t o s e corn syrup (HFCS). The c u r r e n t method o f p r o d u c t i o n o f HFCS i s v i a wet m i l l i n g w h i c h e x p l o i t s the p h y s i c a l p r o p e r t i e s of the whole corn c o n s t i t u e n t s ( o i l , s t a r c h , g l u t e n , and f i b e r ) f o r t h e i r s e p a r a t i o n coupled w i t h enzymatic h y d r o l y s i s o f the starch f r a c t i o n to monosaccharides. The m a j o r i t y o f r e p o r t e d r e s e a r c h a r t i c l e s h a v e d e a l t w i t h low m o l e c u l a r w e i g h t s t a r c h c o n v e r s i o n r e a c t i o n s s u c h as g l u c o s e i s o m e r i z a t i o n and " l i m i t " dextranization (1-4). O n l y a few r e p o r t s have d e a l t w i t h e n z y m a t i c h y d r o l y s i s o f h i g h m o l e c u l a r w e i g h t s t a r c h (5-8) i n s p i t e o f the o b v i o u s i m p o r t a n c e o f t h i s s t e p o f the p r o c e s s ; starch liquefaction. A number o f t e c h n i c a l d i f f i c u l t i e s have prevented q u a n t i t a t i v e m o n i t o r i n g of changes i n t h e m o l e c u l a r p r o p e r t i e s o f s t a r c h undergoing h y d r o l y s i s . Bulk chemical or p h y s i c a l measurements w i l l n o t r e v e a l t h e e s s e n t i a l changes o c c u r r i n g d u r i n g s t a r c h l i q u e f a c t i o n or provide s u f f i c i e n t i n f o r m a t i o n f o r m o d e l i n g the o p e r a t i o n . R e c e n t l y , a s i z e e x c l u s i o n c h r o m a t o g r a p h i c (SEC) a p p a r a t u s has been d e s c r i b e d w h i c h i s c a p a b l e o f p r o v i d i n g the r e q u i r e d i n f o r m a t i o n (9,10) * * i s the aim o f t h i s r e p o r t t o demonstrate t h a t aqueous SEC can be employed t o s t u d y e n z y m a t i c s t a r c h l i q u e f a c t i o n and p r o v i d e a c l e a r p i c t u r e o f the p h y s i c a l and c h e m i c a l e v e n t s o c c u r r i n g d u r i n g t h i s important depolymerization process. t


ROLLINGS E T AL.

Enzymatic

Hydrolysis

Experimental P u r i f i e d c o r n s t a r c h p r e p a r e d by wet m i l l i n g was o b t a i n e d f r o m Sigma C h e m i c a l Company, S t . L o u i s , MO ( l o t no. 68C-0244) and used as s u b s t r a t e . A c o m m e r c i a l g r a d e , e n d o - a c t i n g α-amylase used t o h y d r o l y s e the s t a r c h s u b s t r a t e was a g i f t o f Novo L a b o r a t o r i e s , W i l t o n , CT (Thermamy 1 - h i g h temper a t u r e s t a b l e enzyme). A s t o c k enzyme s o l u t i o n was p r e p a r e d by d i l u t i n g the c o m m e r c i a l g r a d e l i q u i d enzyme ( 1 : 1 9 ) w i t h 0.01 M a c e t a t e b u f f e r pH 6.0 and s t o r e d a t 40°C. T h i s enzyme s o l u t i o n was u s e d f o r t h e h y d r o l y s i s e x p e r i m e n t s as d e s c r i b e d below. A l l c h e m i c a l s used i n t h i s s t u d y were r e a g e n t g r a d e o r b e t t e r . The r e a c t o r u s e d f o r s t a r c h h y d r o l y s i s was a s t a n d a r d two l i t e r Ace G l a s s w a r e , f o u r - p o r t b a t c h reactor. The c e n t r a l p o r t was u s e d t o h o u s e t h e a g i t a t o r s h a f t i n a j a c k e t e d column. The a g i t a t o r was powered by a v a r i a b l e speed m o t o r o p e r a t i n g a t 140 rpm. The t h r e e s i d e p o r t s w e r e u s e d t o h o u s e t h e i n t e r n a l t h e r m i s t o r , a NBS t h e r m o m e t e r , and a t e f l o n p l u g f o r the sampling p o r t . The r e a c t o r was t h e r m o s t a t e d b o t h i n t e r n a l l y and e x t e r n a l l y i n a well-mixed o i l bath. T h i s thermostated system was f o u n d t o a c h i e v e a d e s i r e d t e m p e r a t u r e w i t h i n t h r e e h o u r s and t o m a i n t a i n t h e t e m p e r a t u r e w i t h i n ± 0.3°C c o n t i n u o u s l y t h e r e a f t e r . A schematic r e p r e s e n t a t i o n o f t h e a p p a r a t u s i s shown i n F i g u r e 1 S t a r c h h y d r o l y s a t e s a m p l e s were e x t r a c t e d f r o m t h e b a t c h r e a c t o r a t t i m e d i n t e r v a l s and a s s a y e d by e i t h e r t r a d i t i o n a l end g r o u p a n a l y s i s as d e s c r i b e d p r e v i o u s l y (JLl) o r by SEC. The SEC a p p a r a t u s u s e d i n t h e s e s t u d i e s i s shown i n F i g u r e 2. No s i n g l e c h r o m a t o g r a p h i c r e s i n column i s c a p a b l e o f r e s o l v i n g more t h a n two and one h a l f d e c a d e s i n m o l e c u l a r w e i g h t f o r random c o i l p o l y m e r s (_12). During l i q u e f a c t i o n , s t a r c h m o l e c u l e s range i n m o l e c u l a r s i z e f r o m a p p r o x i m a t e l y 10** dJl/g t o a p p r o x i m a t e l y 10^ dA/g (9). I n o r d e r t o extend the m o l e c u l a r s i z e r a n g e and t h e r e b y o b t a i n a m a x i m a l amount o f m o l e c u l a r i n f o r m a t i o n f r o m each e x p e r i m e n t , a twocolumn SEC s y s t e m was d e v e l o p e d w h i c h i s c a p a b l e o f s e p a r a t i n g 4.5 - 5.0 d e c a d e s i n m o l e c u l a r s i z e f o r water s o l u b l e polymers ( 1 0 ) . T h i s system c o n s i s t s o f a 25.6 cm l o n g S e p h a r o s e CL-6B column c o u p l e d w i t h a 17.85 cm l o n g S e p h a r o s e CL-2B c o l u m n . Each column was 8 mm i n i n s i d e d i a m e t e r . The c h r o m a t o g r a p h i c r e s i n s p a c k e d i n t h e s e columns were f r a c t i o n a t e d by a wet s i e v i n g t e c h n i q u e t o o b t a i n b e a d s o f


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Variable Speed Motor

Oil Bath

Figure 1.

Batch Reactor

Schematic diagram of batch reactor.


21.

ROLLINGS E T A L .

Enzymatic

447

Hydrolysis

w

Positive Displacement Pump

Pulse Dampner

Solvent Filter Conical Flask Solvent Reservoir

Rheodyne Loop Injector

Chromatographic Column Discharge

Chart Recorder

Differential Refractometer Waters 401

Figure 2.

Size exclusion chromatographic (SEC) apparatus.

American Chemical Society Library 1155 16th St., N-W.

Blanch et al.; Foundations of Biochemical Engineering Washington, U.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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40-60 μπι i n s i z e ( 9 0 . T h i s was done t o i n c r e a s e t h e f r a c t i o n a t i o n e f f i c i e n c y t o a p p r o x i m a t e l y 3000 t h e o r e t i c a l p l a t e s p e r m e t e r and s h o r t e n t h e e l u t i o n t i m e t o l e s s t h a n two h o u r s . The e l u e n t and s t a r c h s o l v e n t was 0.5 Ν NaOH. A constant eluent flow r a t e o f 6.5 ml p e r h o u r was m a i n t a i n e d u s i n g a s l o f l o w m i n i pump ( M i l t o n Roy: L a b o r a t o r y D a t a C o n t r o l , R i v i e r a Beach, F L ) . The SEC columns were c a l i b r a t e d u s i n g n a r r o w m o l e c u l a r weight d i s t r i b u t i o n water s o l u b l e polymer s t a n d a r d s ; s o d i u m p o l y s t y r e n e s u l f o n a t e , NaPSS ( P r e s s u r e C h e m i c a l Company, P i t t s b u r g h , PA) and dextrans (Pharmacia Fine Chemicals, Piscataway, NJ). The u n i v e r s a l c a l i b r a t i o n c u r v e p r o p o s e d by G r u b i s i c e t a l . (1J3) was f o u n d n o t t o be a p p l i c a b l e f o r t h e s e polymer standards. The a l t e r n a t i v e c a l i b r a t i o n p r o c e d u r e o f C o l l and P r u s i n o w s k i (14) w h i c h i n c o r p o r a t e s e x c l u d e d volume e f f e c t s i n t h e n u m e r i c a l v a l u e o f t h e F l o r y p a r a m e t e r was f o u n d t o be a p p l i c a b l e f o r t h i s s y s t e m ( 1 5 ) . The c a l i b r a t i o n c u r v e f o r t h e two-column n e t w o r k s y s t e m d e s c r i b e d above i s shown i n F i g u r e 3. The K^y p a r a m e t e r u s e d on the a b s c i s s a o f F i g u r e 3 i s a n o r m a l i z e d e l u t i o n volume p a r a m e t e r d e f i n e d by E q u a t i o n 1, V AV

V

- V t

- V

K

U

ο

where V i s t h e peak r e t e n t i o n volume o f t h e s a m p l e , V i s t h e v o i d volume o f t h e c o l u m n , and V i s the t o t a l i n t e r s t i t i a l volume o f t h e c o l u m n . e

Q

t

R e s u l t s and

Discussion

In o r d e r t o d e s c r i b e the k i n e t i c s o f enzymatic s t a r c h d e p o l y m e r i z a t i o n , i n f o r m a t i o n on r e a c t i o n r a t e , r e a c t i o n e x t e n t , and p r o d u c t d i s t r i b u t i o n p r o f i l e s are r e q u i r e d . T r a d i t i o n a l end-group ana l y s i s can be u s e d t o a l i m i t e d e x t e n t i n t h e f i r s t two a r e a s , b u t w i l l n o t p r o v i d e i n f o r m a t i o n a b o u t the l a s t important s u b j e c t . H e n c e , SEC p r o f i l e s can p r o v i d e s u f f i c i e n t i n s i g h t i n t o t h e mechanism of starch degradation. E f f e c t o f T e m p e r a t u r e on R e a c t i o n R a t e . The i n i t i a l r a t e o f s t a r c h h y d r o l y s i s was i n v e s t i g a t e d a t f i v e t e m p e r a t u r e s (40°C, 57.5°C, 67.5°C, 80°C and 95°C) u s i n g t h r e e d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s ( 0 . 4 4 % , 0.88%, and 1.76% w t / v o l ) by


21.

ROLLINGS E T A L .

Enzymatic

τ

Figure 3.

449

Hydrolysis

1

r——

1

r

C-P calibration curve of SEC column network.


450

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m o n i t o r i n g the p r o d u c t i o n of r e d u c i n g sugars ( d e x t r o s e e q u i v a l e n c e , D.E.). At each i s o t h e r m , the i n i t i a l r a t e d a t a were shown t o f o l l o w M i c h a e l i s - M e n t e n k i n e t i c s (11) c o n s i s t e n t w i t h t h e o b s e r v a t i o n s o f p r e v i o u s r e p o r t s (.16,12)· Hence, L i n e w e a v e r - B u r k p l o t s c o u l d be g e n e r a t e d f r o m the d a t a y i e l d i n g v a l u e s o f a p p a r e n t maximum i n i t i a l v e l o c i t i e s a t e a c h o f the t e m p e r a t u r e s . This i n f o r m a t i o n i s p l o t t e d i n F i g u r e 4 w i t h an assumed A r r h e n i u s dependency, which f o r t h i s system c l e a r l y does n o t e x i s t . The s a l i e n t f e a t u r e s o f t h i s study are that there e x i s t s n e a r l y a three f o l d o r d e r o f m a g n i t u d e i n c r e a s e i n t h e a p p a r e n t maximum v e l o c i t y as t h e s t a r c h s u b s t r a t e i s m o d i f i e d due t o the g e l a t i n i z a t i o n phenomena. A t t h e h i g h e s t tem p e r a t u r e t e s t e d , the r a t e o f t h e r e a c t i o n d e c r e a s e s . T h i s i s due t o t h e r m a l d e a c t i v a t i o n o f t h e enzyme. T h i s l a r g e i n c r e a s e i n the r e a c t i o n r a t e i n d i c a t e s t h a t t h e o v e r r i d i n g e f f e c t i n v o l v e s the p h y s i c a l s t a t e o f the s t a r c h g r a n u l e . Non-gelatinized s t a r c h i s h i g h l y c r y s t a l l i n e (18) and t h u s t h e s u s c e p t i b i l i t y o f s t a r c h t o be e n z y m a t i c a l l y h y d r o l y z e d u n d e r t h e s e c o n d i t i o n s i s much l o w e r t h a n f o r s t a r c h g r a n u l e s t h a t have been d i s r u p t e d by h e a t i n g i n aqueous s u s p e n s i o n . Through t h e p r o c e s s o f g e l a t i n i z a t i o n , the o r i g i n a l i n t e r n a l o r d e r i n g of the s t a r c h m o l e c u l e s w i t h i n the g r a n u l e s i s d i s r u p t e d , the g r a n u l e i m b i b e s l a r g e amounts o f w a t e r and s w e l l s . E v e n t u a l l y due t o combined a c t i o n o f s h e a r i n g w i t h i n t h e r e a c t o r and o s m o t i c s w e l l i n g , granule i n t e g r i t y i s completely lost. L a r g e s t a r c h m o l e c u l e s may t h e n r e a s s o c i a t e ( r e t r o grade) producing a g e l m a t r i x . I t c a n n o t be assumed t h a t t h e p r o g e s s o f t h e r e a c t i o n w i l l f o l l o w the same s e q u e n c e i f t h e s u b s t r a t e i s i n v a r i o u s physical states. I t i s therefore of i n t e r e s t to f o l l o w the m o l e c u l a r w e i g h t d i s t r i b u t i o n p r o f i l e s d u r i n g the c o u r s e of the r e a c t i o n at v a r i o u s s t a t e s of macromolecular aggregation. The aqueous SEC a p p a r a t u s d e s c r i b e d above was s p e c i f i c a l l y d e v e l o p e d for t h i s purpose. T h i s t e c h n i q u e was shown t o be a p p r o x i m a t e l y 10^ t i m e s more a c c u r a t e t h a n r e d u c i n g s u g a r measurements f o r low d e g r e e o f c o n v e r s i o n starch h y d r o l y s i s reactions (10). E f f e c t o f T e m p e r a t u r e on H y d r o l y s i s E x t e n t and P r o d u c t D i s t r i b u t i o n . I t was shown above t h a t s l o w i n i t i a l r a t e of r e a c t i o n i s c h a r a c t e r i s t i c of opera t i n g a t low t e m p e r a t u r e s ( b e l o w t h e g e l a t i n i z a t i o n range of s t a r c h ) whereas r a p i d i n i t i a l r a t e s are


ROLLINGS E T A L .

Enzymatic

Hydrolysis

TEMPERATURE ( Ο β

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Figure 4.

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A rrhenius plot of • max app.


BIOCHEMICAL

ENGINEERING

o b s e r v e d a t h i g h t e m p e r a t u r e s ( a b o v e the g e l a t i n i z a t i o n of starch). However, t h e d i s t r i b u t i o n s o f m a c r o m o l e c u l a r components d u r i n g h y d r o l y s i s a t v a r i o u s t e m p e r a t u r e s have n o t b e e n r e p o r t e d and a r e thus o f i n t e r e s t . SEC p r o f i l e s o f h y d r o l y s i s p r o d u c t s o f enzymet r e a t e d s t a r c h as a f u n c t i o n o f r e a c t i o n t i m e a t 60°C and 80°C a r e shown i n F i g u r e s 5 and 6 r e s p e c tively. I n b o t h c a s e s , a common enzyme dose o f one ml o f t h e 1:19 s t o c k Novo Thermamyl s o l u t i o n was m i x e d w i t h a s l u r r y o f 15 gms s t a r c h d r y b a s i s i n 75 ml w a t e r and added t o t h e r e a c t o r . The f i n a l volume was 1500 m l . A t 60°C, b e l o w t h e g e l a t i n i z a t i o n ( m e l t i n g ) range o f the g r a n u l e s , the p r o d u c t d i s t r i b u t i o n i s e x t r e m e l y non-random ( s e e F i g u r e 5 ) . Only d u r i n g t h e i n i t i a l s t a g e s o f r e a c t i o n i s any s i g n i f i c a n t amount o f i n t e r m e d i a t e m o l e c u l a r s i z e p r o d u c t s ( i . e . , b e t w e e n h y d r o d y n a m i c volume 10^ and 10^ dl/mole) detected. Throughout the time course of l i q u e f a c t i o n , the m a j o r i t y o f t h e m o l e c u l a r s p e c i e s present are e i t h e r very h i g h i n molecular weight ( e x c l u d e d from t h e SEC column) o r low m o l e c u l a r w e i g h t end p r o d u c t s . Low m o l e c u l a r w e i g h t p r o d u c t s c o n t i n u o u s l y accumulate w h i l e h i g h m o l e c u l a r weight m a t e r i a l s d i s a p p e a r w i t h o u t d e t e c t i o n o f any s i g n i f i c a n t amount o f i n t e r m e d i a t e m o l e c u l a r w e i g h t material. T h i s s i t u a t i o n c o u l d r e s u l t i f t h e enzyme i s a l l o w e d t o a c t o n l y on r e l a t i v e l y s h o r t , e x p o s e d c h a i n s of s t a r c h present i n the c r y s t a l l i n e m a t r i x o f the g r a n u l e , o r i f l o n g e r c h a i n m o l e c u l e s f r e e d f r o m t h e g r a n u l e a r e more s u s c e p t i b l e t o enzyme h y d r o l y s i s than the molecules a s s o c i a t e d w i t h the g r a n u l e and a r e t h e r e f o r e p r e f e r e n t i a l l y d e g r a d e d . A l t e r n a t i v e l y t h i s s i t u a t i o n c o u l d a l s o a r i s e i f the a v a i l a b l e s t a r c h i s s i m p l y o v e r l o a d e d w i t h enzyme. Other authors suggest that p r e f e r e n t i a l " p i t t i n g " may o c c u r due t o d i f f e r i n g d e g r e e s i n s u b s t r a t e susceptibility. The r e a c t i o n u n d e r t h e s e c o n d i t i o n s does procède t o c o m p l e t i o n a l t h o u g h r e q u i r i n g a p p r o x i m a t e l y ten hours f o r the c o n v e r s i o n . Above t h e g e l a t i n i z a t i o n r a n g e (80°C, see F i g u r e 6) t h e p r o d u c t d i s t r i b u t i o n d u r i n g r e a c t i o n i s c o n s i d e r a b l y d i f f e r e n t t h a n 60°C. A g a i n , the d i s t r i b u t i o n o f m o l e c u l a r s i z e s i s n o t random. D u r i n g t h e e a r l y s t a g e s o f the r e a c t i o n , i n a d d i t i o n t o t h e v e r y h i g h m o l e c u l a r w e i g h t and low m o l e c u l a r weight products, a large f r a c t i o n of intermediate m o l e c u l a r weight d e x t r i n s (between hydrodynamic v a l u e 10^ and 10·* d l / m o l e , see F i g u r e 5) a r e p r e s e n t .


Figure 5.

Chromatograms of starch hydrolyzed at 60°C at indicated times during reaction. Top curve, 17 min: 26 s.


454

BIOCHEMICAL

1

0.0

I

02

I

I

0.4

0.6 K

Figure 6.

I

0.8

ENGINEERING

I

1.0

AV

Chromatograms of starch hydrolyzed at 80°C at indicated times during reaction. Times indicated as min: s.


Enzymatic

ROLLINGS E T A L .

Hydrolysis

A t one and o n e - h a l f m i n u t e s i n t o t h e r e a c t i o n , two d i s c r e t e i n t e r m e d i a t e m o l e c u l a r weight peaks a r e detected at K v a l u e s o f 0.25 and 0.60. These values correspond t o approximate m o l e c u l a r weights o f 5 χ 10^ and 10^ r e s p e c t i v e l y (9). Throughout the r e m a i n i n g time course o f t h e r e a c t i o n these peaks p e r s i s t , t h o u g h t h e i r K values increase s l i g h t l y ( t o 0.275 and 0.65 r e s p e c t i v e l y ) w i t h i n c r e a s i n g time c h a r a c t e r i s t i c o f a r e d u c t i o n i n t h e i r molecular weight v a l u e s . This i s apparently due t o g r a d u a l e n d - w i s e d e p o l y m e r i z a t i o n o f t h e s e intermediates. T h i s i n d i c a t e s t h a t under these r e a c t i o n c o n d i t i o n s , c e r t a i n intermediate products a r e more r e s i s t a n t t o e n z y m a t i c a t t a c k , and t h e r e f o r e a p p e a r as d i s c r e t e c h r o m a t o g r a p h i c p e a k s . D i s c r e t e peaks c o u l d r e s u l t i f c e r t a i n m o l e c u l a r s i z e starch molecules are e i t h e r p r e f e r e n t i a l l y produced (as b y - p r o d u c t s ) o r a r e r e s i s t a n t t o e n z y m a t i c a t t a c k , and t h e r e f o r e a p p e a r as d i s c r e t e chromatographic peaks. P r e v i o u s r e p o r t s o f such i n t e r m e d i a t e s d u r i n g s t a r c h l i q u e f a c t i o n have n o t been f o u n d . A V

A V

No f u r t h e r changes i n t h e c h r o m a t o g r a p h i c t r a c i n g s a r e seen beyond e l e v e n and o n e - h a l f m i n u t e s a t 80°C, w h i c h shows t h a t t h e r m a l d e a c t i v a t i o n o f t h e enzyme h a s s t o p p e d t h e p r o g r e s s o f t h e r e a c t i o n . The r e a c t i o n does n o t p r o c e e d t o c o m p l e t i o n u n d e r these c o n d i t i o n s . The f i n a l p r o d u c t d i s t r i b u t i o n o f a s t a r c h s o l u t i o n h y d r o l y z e d a t 95°C u n d e r t h e same c o n d i t i o n s as t h e o t h e r two i s o t h e r m s was d e t e r m i n e d and i s shown t o g e t h e r w i t h t h e s i m i l a r d a t a a t 80°C, 65°C, and 60°C i n F i g u r e 7. F o u r d i s t i n c t peaks a r e o b s e r v e d : one a t t h e low m o l e c u l a r w e i g h t l i m i t , and two i n t e r m e d i a t e m o l e c u l a r w e i g h t p e a k s a t K^y v a l u e s o f 0.2 and 0.6 r e s p e c t i v e l y . This i s the same t y p e o f c h r o m a t o g r a p h i c b e h a v i o r o b s e r v e d d u r i n g t h e i n i t i a l s t a g e s o f r e a c t i o n a t 80°C. Under a l l t h e r m a l s t a t e s t e s t e d , a non-random distribution exists. E f f e c t o f S t a r c h R e c r y s t a l l i z a t i o n on H y d r o l y s i s E x t e n t , P r o d u c t D i s t r i b u t i o n , and R e a c t i o n Rates. Since retrograded ( r e c r y s t a l l i z e d ) starch i s t h o u g h t t o be more r e s i s t a n t t o e n z y m a t i c d e p o l y m e r i z a t i o n t h a n u n r e t r o g r a d e d s t a r c h (.22,22), i t i s o f i n t e r e s t t o compare t h e h y d r o l y s i s o f r e t r o g r a d e d starch to non-retrograded starch. Extremely r e t r o g r a d e d s t a r c h was p r e p a r e d by f r e e z e - d r y i n g p r e g e l a t i n i z e d s t a r c h , and b a l l - m i l l i n g t h i s m a t e r i a l t o a


456

BIOCHEMICAL

Figure 7.

ENGINEERING

Comparison of final product distribution of starch hydrolyzed at 95°C, 80°C, 65°C, and60°C.


Enzymatic

ROLLINGS E T AL.

Hydrolysis

f i n e powder. T h i s m a t e r i a l was e n z y m a t i c a l l y h y d r o l y z e d a t 80°C, and t h e m o l e c u l a r s i z e d i s t r i b u t i o n was d e t e r m i n e d by s i z e e x c l u s i o n c h r o m a t o graphy. The c h r o m a t o g r a p h i c p r o f i l e s a r e shown i n F i g u r e 8. The c h r o m a t o g r a p h i c p r o f i l e o f unreacted, p r e g e l a t i n i z e d , f r e e z e - d r i e d , b a l l m i l l e d s t a r c h ("0" t i m e c h r o m a t o g r a m ) shows t h a t s i g n i f i c a n t d e p o l y m e r i z a t i o n o c c u r r e d d u r i n g the b a l l - m i l l i n g operation. D u r i n g the course o f enzymatic d e p o l y m e r i z a t i o n of t h i s r e c r y s t a l l i z e d s t a r c h , f o u r d i s t i n c t p e a k s a r e p r e s e n t : one n e a r t h e e x c l u s i o n l i m i t o f t h e column s e t , one a t the end p r o d u c t s , and two i n t e r m e d i a t e p e a k s . These i n t e r m e d i a t e p e a k s a r e d e t e c t a b l e as e a r l y as two and o n e - h a l f m i n u t e s i n t o t h e r e a c t i o n . The p e a k s a p p e a r e d a t K y v a l u e s o f a p p r o x i m a t e l y 0.25 and 0.6, t h e same v a l u e s o b s e r v e d when n o n - p r e g e l a t i n i z e d s t a r c h was r e a c t e d a t 80°C. These values for t h e two i n t e r m e d i a t e p e a k s i n c r e a s e d s l i g h t l y w i t h i n c r e a s i n g r e a c t i o n time, a g a i n c o n s i s t e n t w i t h the r e s u l t s shown a t 80°C f o r n o n - p r e g e l a t i n i z e d s t a r c h . Thus t h e r e s i s t a n t , i n t e r m e d i a t e m o l e c u l a r s i z e s t a r c h p r o d u c t s formed a f t e r s e v e r e rétrogradation d u r i n g f r e e z i n g are q u i t e s i m i l a r t o the groups formed when a n o n - p r e g e l a t i n i z e d s t a r c h i s enzyma t i c a l l y d e p o l y m e r i z e d u n d e r t h e same c o n d i t i o n s . No change i n t h e c h r o m a t o g r a p h i c p r o f i l e s i s d e t e c t e d a f t e r e l e v e n m i n u t e s and the r e a c t i o n does not proceed to completion. A

Conclusion The p r e s e n t i n v e s t i g a t i o n has p r o v i d e d new i n s i g h t i n t o the p h y s i c a l e v e n t s o c c u r r i n g d u r i n g enzymatic s t a r c h d e p o l y m e r i z a t i o n . Degradation of s t a r c h u n d e r t h e c o n d i t i o n s t e s t e d does n o t p r o c e e d by a random p r o c e s s . Granule s t r u c t u r e a f f e c t s the m o l e c u l a r weight d i s t r i b u t i o n p r o f i l e s of the s t a r c h hydrolysates. S t a r c h h y d r o l y s a t e s produced at temperatures below the g e l a t i n i z a t i o n range o f the s u b s t r a t e a r e composed m a i n l y o f h i g h m o l e c u l a r w e i g h t m a t e r i a l o r low m o l e c u l a r w e i g h t end p r o d u c t s . A t 80°C, above t h e m e l t i n g r a n g e o f t h e g r a n u l e s , a l a r g e amount o f i n t e r m e d i a t e m o l e c u l a r s i z e compon e n t s e x i s t i n a d d i t i o n t o t h e h i g h and low m o l e c u l a r weight groups. These i n t e r m e d i a t e p r o d u c t s a r e a p p r o x i m a t e l y 5 χ 10^ and 10^ i n m o l e c u l a r w e i g h t and a r e t h e same s i z e components s e e n i n t h e h y d r o l y s i s o f h i g h l y r e c r y s t a l l i z e d s t a r c h r e a c t e d at the same i s o t h e r m . T h i s suggests t h a t the r e c r y s t a l l i -


Figure 8.

Chromatograms of pregelatinized starch hydrolyzed at 80°C at indicated times (min: s) during reaction.


21.

ROLLINGS

ET AL.

Enzymatic

Hydrolysis

z a t i o n p r o c e s s w i l l o c c u r even a t h i g h t e m p e r a t u r e operation. Although the i n i t i a l r a t e o f the r e a c t i o n i s g r e a t e s t a t t e m p e r a t u r e s above t h e g e l a t i n i z a t i o n range o f the s u b s t r a t e , the f i n a l e x t e n t o f con v e r s i o n i s g r e a t e s t a t low t e m p e r a t u r e o p e r a t i o n f o r a common amount o f enzyme l o a d i n g . This s i t u a t i o n e x i s t s due t o t h e r m a l d e a c t i v a t i o n o f t h e catalyst. Optimal operation of a starch l i q u e f a c t i o n o p e r a t i o n must a c c o u n t f o r b o t h t h e r a t e o f i n c r e a s e d s u b s t r a t e s u s c e p t i b i l i t y c a u s e d by t h e d i s r u p t i o n o f the i n t e r n a l macromolecular o r d e r i n g w i t h i n t h e g r a n u l e and t h e l o s s o f e n z y m a t i c a c t i v i t y due t o t h e r m a l d e a c t i v a t i o n . A p h y s i c a l model o f s t a r c h g e l a t i n i z a t i o n , rétrogradation, and l i q u e f a c t i o n c a n be p r e s e n t e d from these r e s u l t s . I t i s c l e a r that molecular r e c r y s t a l l i z a t i o n v i a rétrogradation p r o c e s s w i l l occur quite r a p i d l y f o r p r e g e l a t i n i z e d starch. I t i s t h e r e f o r e not p o s s i b l e to study the enzymatic hydrolysis of starch without considering r e t r o gradation e f f e c t s . The p h y s i c a l p i c t u r e f o r t h e o v e r a l l p r o c e s s i s summarized i n F i g u r e 9. The i n i t i a l p o p u l a t i o n o f s t a r c h g r a n u l e s i s suspended i n aqueous s o l u t i o n . When s u b j e c t e d f o r a p e r i o d o f t i m e a t o r above t h e g e l a t i n i z a t i o n t e m p e r a t u r e r a n g e o f t h e m a t e r i a l , some o f t h e s t a r c h g r a n u l e s melt. The m o l e c u l a r components o f t h e g r a n u l e w i l l then r e a s s o c i a t e t o form c r y s t a l l i n e b o d i e s , some o f w h i c h may be more o r l e s s r e s i s t a n t t o enzymatic h y d r o l y s i s than other c r y s t a l types. The enzyme w i l l h y d r o l y z e s u s c e p t i b l e bond l i n k a g e s t o low m o l e c u l a r w e i g h t o l i g o s a c c h a r i d e s as a f u n c t i o n o f t i m e and c o n c e n t r a t i o n .


459


• · ·

·····

Tt

Figure 9.

·

Population of Granules

°

· ° T

,

C

T.t.Cs

+ hydrolyis products

,E(t),t

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Mixture of gelatinized granules, retrograded materials + soluble components

+ hydrolysis products

Mixture of ungelatinized, gelatinized granules, retrograded material + solublecomponents

Physical model of gelatinization, rétrogradation, and liquefaction.

·

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Mixture of ungelatinizedjgelatinized granules + soluble components

ROLLINGS E TA L .

Enzymatic

Hydrolysis

Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15.

16. 17. 18. 19. 20. 21. 22.

23.

Marsh, D.R.; Lee, Y.Y.; Tsao, G.T. B i o t e c h n o l . Bioeng. 1973, 15, 483. Lee, D.D.; Lee, Y.Y.; R e i l l y , P.J.; C o l l i n g s , E.V.; Tsao, G.T. B i o t e c h n o l . Bioeng. 1976, 18, 253. Wight, A.W. Die Starke 1976, 28, 311. John, M. and Dellweg, H. Sept. and Pur. Meth. 1973, 2, 231. Cruz, A. Ph.D. T h e s i s , Princeton U n i v e r s i t y , P r i n c e t o n , NJ, 1976. Henriksnas, H. and Bruun, H. Die Starke 1978, 30, 233. Henriksnas, H. and Lovgren, T. B i o t e c h n o l . Bioeng. 1978, 20, 1303. B o n d e t s k i i , K.M. and Yarovenko, V.L. Bioorgan. Khimiia 1975,1.,614. R o l l i n g s , J.E. Ph.D. T h e s i s , Purdue U n i v e r s i t y , L a f a y e t t e , IN, 1978. R o l l i n g s , J.E.; Bose, Α.; Okos, M.R.; and Tsao, G.T. J. Appl. Polym. S c i . (accepted). R o l l i n g s , J.E. M.S. T h e s i s , Purdue U n i v e r s i t y , L a f a y e t t e , IN, 1978. Yau, W.W.; K i r k l a n d , J . J . ; and Bly, D.D. "Modern Size E x l u s i o n Chromatography" ; John Wiley and Sons, NY, 1979, 97. G r u b i s i c , Z.; Rempp, R.; and Benoit, H. J . Polym. S c i . Part Β 1967, 5, 753. C o l l , H. and Prusinowski, P. J . Polym. S c i . Part Β 1967, 5, 1153. Bose, Α.; R o l l i n g s , J.E.; Caruthers, J.M.; Okos, M.R.; and Tsao, G.T. J. Applied Polym. S c i . (accepted). Rosendal, P.; N i e l s e n , B.H.; and Lange, Ν.Κ. Die Starke 1979, 31, 368. Roels, J.A. and v a n T i l b u r g , R. Die Starke 1979, 31, 338. S t e r l i n g , C. J. Texture Studies 1978, 9, 225. Chabat, J.F.; A l l e n , A.E.; and Hood, L.F. J . Food S c i . 1978, 43, 727. H o l l i n g e r , L.F. and Marchessault, R.H. Biopolymers 1975, 14, 265. Mussulman, W.C. and Wagoner, J.A. Cereal Chem. 1968, 45, 162. Watson, S.A. in "Methods i n Carbonydrate Chemistry, Vo. IV" R.L. W h i s t l e r , ed.;Academic Press, NY, 1964. Boruch, W.M. and P i e r z g a l s k i , T. Die Starke 1979, 31, 149.

R E C E I V E D June 1, 1982


Foundations of Biochemical Engineering - American Chemical Society

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