Enzymes in Biomass Conversion - American Chemical Society

Chapter 30

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Reactions of Glucansucrases in the Biomass Conversion of Sucrose John F. Robyt Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50010

Many different kinds of glucansucrases are elaborated by different species of Leuconostoc and Streptococcus to give different kinds of glucans from sucrose. Mechanistic studies have shown that two different dextransucrases and a mutansucrase form covalent glucosyl and glucanosyl intermediates and that the glucosyl moiety is transferred to the reducing-end of the growing glucanosyl chain. The synthesis of the glucan is terminated when the glucan chain is released by acceptor reactions. Acceptor reactions occur when other carbohydrates besides sucrose are added to the digests. When glucan itself is the acceptor, a branch linkage is formed. Over 50 different carbohydrates are known acceptors. The different acceptors react at different rates and efficiencies. The structures of the acceptor products depend on the structure of the acceptor. The amounts of glucan and acceptor product can be altered by the concentration ratio of acceptor to sucrose. Glucansucrases can, thus, be utilized to produce new products (glucans and oligosaccharides of different structures) from the biomass material, sucrose. S t a r c h , c e l l u l o s e , a n d s u c r o s e a r e abundant, renewable raw m a t e r i a l s t h a t c a n be u s e d i n biomass c o n v e r s i o n s . Glucansucrases a r e enzymes t h a t c o n v e r t s u c r o s e i n t o g l u c a n s , and o n t h e a d d i t i o n of other carbohydrates t o the sucrose d i g e s t s , the glucose moiety o f sucrose i s t r a n s f e r r e d to the carbohydrates to give o l i g o s a c c h a r i d e acceptor products. G l u c a n s u c r a s e s , t h u s , a r e i m p o r t a n t enzymes i n t h e biomass c o n v e r s i o n o f s u c r o s e i n t o new p r o d u c t s , e i t h e r p o l y saccharides or oligosaccharides. G l u c a n s u c r a s e s a r e e l a b o r a t e d by Lactobacilli (Leuconostoc and Streptococcus s p e c i e s ) and c a t a l y z e t h e s y n t h e s i s o f g l u c a n s from sucrose. I n 1954, J e a n e s et al. (2) s u r v e y e d 96 L. mesenteroides s p e c i e s t h a t p r o d u c e g l u c a n s from s u c r o s e and f o u n d t h a t t h e g l u c a n s

0097-6156/91/0460-0394$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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had a wide v a r i e t y o f p h y s i c a l and c h e m i c a l p r o p e r t i e s . Some o f the s p e c i e s p r o d u c e two k i n d s o f g l u c a n s t h a t c a n be s e p a r a t e d by d i f f e r e n t i a l alcohol p r e c i p i t a t i o n (2). L. mes enteroides B-512F p r o d u c e s o n l y one g l u c a n , the c l a s s i c a l d e x t r a n t h a t has 95% a-l-+6 l i n k a g e s i n the main c h a i n s w i t h 5% a-l->3 b r a n c h l i n k a g e s . The b r a n c h c h a i n s c o n s i s t o f b o t h s i n g l e g l u c o s e r e s i d u e s and l o n g a-l->6 l i n k e d c h a i n s w i t h many g l u c o s e r e s i d u e s . To d a t e , t h i s i s the o n l y c o m m e r c i a l l y p r o d u c e d g l u c a n from s u c r o s e . The r e s u l t i n g d e x t r a n i s o f v e r y h i g h m o l e c u l a r w e i g h t (> 5 χ 10 ) . I t s e r v e s as the s t a r t i n g m a t e r i a l f o r the e p i c h l o r o h y d r i n c r o s s l i n k e d , g e l - f i l t r a t i o n m a t e r i a l , Sephadex. Low m o l e c u l a r weight d e x t r a n (5-10 χ 10*) p r o d u c e d by c o n t r o l l e d a c i d h y d r o l y s i s and a l c o h o l f r a c t i o n a t i o n i s u s e d as a b l o o d - p l a s m a s u b s t i t u t e (3) (see F i g . 1A f o r the s t r u c t u r e ) . A low m o l e c u l a r w e i g h t d e x t r a n s u l f a t e w i l l s e r v e as a s u b s t i t u t e f o r the a n t i c o a g u l a n t p o l y s a c c h a r i d e , heparin. Streptococcus mutans p r o d u c e s two g l u c a n s u c r a s e s , d e x t r a n s u c r a s e and mutansucrase. D e x t r a n s u c r a s e s y n t h e s i z e s a w a t e r s o l u b l e d e x t r a n t h a t i s r e l a t i v e l y h i g h l y b r a n c h e d w i t h 64% a-1->6 l i n k a g e s i n the main c h a i n s and 36% a-1->3 b r a n c h l i n k a g e s ( 4 ) . The b r a n c h c h a i n s c o n s i s t p r i m a r i l y of s i n g l e glucose residues attached to every other g l u c o s e r e s i d u e i n the main c h a i n s (4) . There a r e some l o n g b r a n c h c h a i n s t h a t a l s o have s i n g l e g l u c o s e u n i t s a t t a c h e d by a-l-+3 b r a n c h l i n k a g e s t o e v e r y o t h e r g l u c o s e r e s i d u e i n the c h a i n . T h i s t y p e o f s t r u c t u r e r e p r e s e n t s a b i f u r c a t e d , a l t e r n a t i n g comb polymer and hence d i f f e r s s i g n i f i c a n t l y i n s t r u c t u r e from B-512F d e x t r a n . It is r e s i s t a n t to endo-dextranase h y d r o l y s i s . Mutansucrase s y n t h e s i z e s a g l u c a n t h a t has >93% a-l-+3 l i n k a g e s . T h i s g l u c a n i s e x t r e m e l y i n s o l u b l e (< 0.01 mg/mL) . Both o f t h e s e g l u c a n s a r e s y n t h e s i z e d i n the o r a l c a v i t y and go t o make-up d e n t a l p l a q u e . The two enzymes have b e e n s e p a r a t e d (5) and the s o l u b l e a l t e r n a t i n g comb d e x t r a n might have some u s e s as a d e x t r a n a s e - r e s i s t a n t g l u c a n , w i t h p r o p e r t i e s d i f f e r e n t from t h o s e o f B-512F d e x t r a n . 7

L. mesenteroides Β-742 p r o d u c e s a d e x t r a n s u c r a s e t h a t s y n t h e s i z e s the most h i g h l y b r a n c h e d d e x t r a n known. I t has 50% a-l-+6 l i n k ages i n the main c h a i n s and 50% a-l-+3 b r a n c h l i n k a g e s . I t s s t r u c t u r e i s s i m i l a r t o t h a t o f S. mutans d e x t r a n w i t h the b r a n c h c h a i n s b e i n g p r i m a r i l y s i n g l e glucose residues. I t d i f f e r s from the S. mutans d e x t r a n i n t h a t e v e r y g l u c o s e r e s i d u e i n the a-1-^6 l i n k e d main c h a i n s has a t t a c h e d t o i t an a-l-*3 l i n k e d g l u c o s e r e s i d u e . T h e r e a r e a l s o some l o n g b r a n c h c h a i n s as w e l l . This structure i s a bifurcated, r e g u l a r comb polymer. These t y p e s o f p o l y m e r s a r e c a l l e d comb p o l y m e r s b e c a u s e the s i n g l e g l u c o s e b r a n c h e s a r e "hung" o f f o f the main c h a i n s as t e e t h on a comb. The B-742 s t r u c t u r e i s c o n s i d e r e d r e g u l a r i n t h a t e v e r y g l u c o s e r e s i d u e i n t h e main c h a i n s a r e b r a n c h e d , whereas the S. mutans comb polymer has "gaps" i n t h e t e e t h as o n l y e v e r y o t h e r g l u c o s e r e s i d u e i n the main c h a i n s a r e b r a n c h e d (see F i g . IB and ID f o r a c o m p a r i s o n o f the two s t r u c t u r e s ) . This d e x t r a n i s a l s o r e s i s t a n t to endo-dextranase h y d r o l y s i s . The h i g h d e g r e e o f b r a n c h i n g o f t h e s e d e x t r a n s a l s o make them v e r y w a t e r soluble.


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Fig. 1

S t r u c t u r e s o f some g l u c a n s

s y n t h e s i z e d by

glucansucrases

(A) d e x t r a n (95% α-1-+6 l i n k a g e s and 5% cr-l-+3 b r a n c h l i n k a g e s ) s y n t h e s i z e d by L e u c o n o s t o c m e s e n t e r o i d e s B-512F d e x t a n s u c r a s e ; (B) a l t e r n a t i n g comb d e x t r a n (65% α-1-+6 l i n k a g e s and 35% a-l-*3 b r a n c h l i n k a g e s ) s y n t h e s i z e d by S t r e p t o c o c c u s mutans dextransucrase; (C) mutan sucrase;

(>93%

a-l-*3 l i n k a g e s )

s y n t h e s i z e d by

S.

mutans

mutan-

(D) r e g u l a r comb d e x t r a n (50% a-l-*6 l i n k a g e s and 50% a-l-*3 b r a n c h l i n k a g e s ) s y n t h e s i z e d by L. mesenteroides B-742 d e x t r a n s u c r a s e ; (E) a l t e r n a n (50% a-l->6 l i n k a g e s a l t e r n a t i n g w i t h 40% a-l->3 l i n k ages and 10% a-l-*3 b r a n c h l i n k a g e s ) s y n t h e s i z e d by L. mesenteroides Β-1355 a l t e r n a n s u c r a s e ; (F) amylose (100% α-l->4 l i n k a g e s ) s y n t h e s i z e d by Neisseria s p e c i e s amylosucrase;

perflava

(G) a m y l o p e c t i n (95% a-l->4 l i n k a g e s w i t h 5% a-l->6 b r a n c h l i n k a g e s ) s y n t h e s i z e d by N. perflava s p e c i e s amylosucrase. W h e r e r e p r e s e n t s a glucopyranosyl residue g l u c o s e r e s i d u e by an a-l->6 l i n k a g e ; p y r a n o s y l r e s i d u e attached to another glucose l i n k a g e ; and Q-O represents a glucopyranosyl a n o t h e r g l u c o s e r e s i d u e by an a-1->4 l i n k a g e .

attached to another represents a gluco r e s i d u e by an a-1-*3 residue attached to


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L. mesenteroides B-1355 p r o d u c e s an i n t e r e s t i n g g l u c a n from s u c r o s e t h a t i s c a l l e d a l t e r n a n (6) b e c a u s e i t has a l t e r n a t i n g a-l-+6 and a-1->3 l i n k a g e s i n t h e main c h a i n s . I t a l s o has a s i g n i f i c a n t number (11%) o f a-1->3 b r a n c h l i n k a g e s . Because o f t h e u n u s u a l a l t e r n a t i n g s t r u c t u r e , a l t e r n a n i s a l s o q u i t e r e s i s t a n t t o endod e x t r a n a s e h y d r o l y s i s and has a v e r y d i f f e r e n t p h y s i c a l appearance from t h a t o f B-512F d e x t r a n when p r e c i p i t a t e d b y a l c o h o l . B-512F d e x t r a n appears as a t r a n s l u c e n t , g e l - l i k e m a t e r i a l , and a l t e r n a n i s a dense w h i t e , o p a q u e - m a t e r i a l (3,6). We have r e c e n t l y i s o l a t e d a s e r i e s o f Neisseria perflava s p e c i e s from d e n t a l p l a q u e t h a t s y n t h e s i z e g l u c a n s w i t h d i f f e r e n t i o d i n e s t a i n i n g p r o p e r t i e s from s u c r o s e . Some s t a i n a v e r y d a r k b l u e - b l a c k , i n d i c a t i n g an a m y l o s e - l i k e s t r u c t u r e w i t h a h i g h c o n t e n t o f con s e c u t i v e a-l-+4 l i n k a g e s ; w h i l e o t h e r s s t a i n p u r p l e , i n d i c a t i n g an a m y l o p e c t i n s t r u c t u r e w i t h a-l->4 l i n k a g e s i n t h e main c h a i n s and al-*6 b r a n c h l i n k a g e s ; and s t i l l o t h e r s s t a i n red-brown, i n d i c a t i n g a more h i g h l y b r a n c h e d , g l y c o g e n - l i k e s t r u c t u r e . F u r t h e r work on t h e enzymes t h a t s y n t h e s i z e t h e s e p o l y s a c c h a r i d e s c o u l d p r o d u c e s t a r c h o r g l y c o g e n p r o d u c t s from s u c r o s e . Three o f t h e g l u c a n s u c r a s e s , L. mesenteroides B-512F d e x t r a n s u c r a s e and S. mutans d e x t r a n s u c r a s e and mutansucrase have b e e n p u r i f i e d ( 5 , 7 - 9 ) , and t h e i r mechanism and s p e c i f i c i t y o f a c t i o n s t u d i e d by p u l s e and chase l a b e l i n g t e c h n i q u e s w i t h C-sucrose (5,10-13). I t h a s been shown t h a t t h e s e enzymes form c o v a l e n t g l u c o s y l and g l u c a n o s y l i n t e r m e d i a t e s d u r i n g s y n t h e s i s and t h e g l u c o s e u n i t i s added t o t h e r e d u c i n g end o f t h e g r o w i n g g l u c a n c h a i n (5,20). F o r L. mesenteroides B-512F d e x t r a n s u c r a s e , the proposed mechanism f o r t h e s y n t h e s i s o f a sequence o f a-1-*6 l i n k a g e s i n v o l v e s two n u c l e o p h i l e s a t t h e a c t i v e s i t e t h a t a t t a c k s u c r o s e and d i s p l a c e f r u c t o s e t o g i v e two / J - g l u c o s y l enzyme i n t e r m e d i a t e s . The C-6-0H o f one o f t h e g l u c o s y l r e s i d u e s i s o r i e n t e d t o a t t a c k C - l o f t h e o t h e r t o form an a-l->6 l i n k a g e . The n u c l e o p h i l e t h a t i s r e l e a s e d a t t a c k s a n o t h e r s u c r o s e f o r m i n g a new e n z y m e - g l u c o s y l i n t e r m e d i a t e . The C-6OH o f t h i s new g l u c o s y l i n t e r m e d i a t e a t t a c k s C - l o f t h e i s o m a l t o s y l u n i t f o r m i n g i s o m a l t o t r i o s y l enzyme i n t e r m e d i a t e and i n e f f e c t i s added t o t h e r e d u c i n g end o f t h e growing d e x t r a n c h a i n . The p r o c e s s c o n t i n u e s i n w h i c h t h e g l u c o s y l and t h e growing d e x t r a n o s y l c h a i n a r e a l t e r n a t e l y t r a n s f e r r e d between the two n u c l e o p h i l e s as t h e d e x t r a n chain i s elongated ( s e e F i g . 2A). A s i m i l a r mechanism c a n be p o s t u l a t e d f o r mutansucrase, b u t w i t h t h e g l u c o s y l u n i t s b e i n g o r i e n t e d so t h a t C-3-0H a t t a c k s C - l o f t h e growing mutan c h a i n t o g i v e a-l->3 i n s t e a d o f a-1-^6 l i n k a g e s ( s e e F i g . 2B). For the mechanism o f a l t e r n a n s u c r a s e , i t c a n be p o s t u l a t e d t h a t t h e two n u c l e o p h i l e s , X and Y, o r i e n t t h e g l u c o s y l u n i t s so t h a t X p l a c e s t h e C-6-0H i n p o s i t i o n t o a t t a c k C - l and form an α-l->6 l i n k a g e and Y p l a c e s t h e C-3-0H i n p o s i t i o n t o a t t a c k C - l and form an a-l-»3 l i n k age. I n t h i s manner t h e a l t e r n a t i n g a-l-»6, a-l-*3 s t r u c t u r e o f a l t e r n a n c a n be s y n t h e s i z e d ( s e e F i g . 2C) . I t s h o u l d be n o t e d t h a t i n D - g l u c o p y r a n o s e , t h e c o n f i g u r a t i o n o f b o t h t h e C-6-0H and t h e C-3OH a r e b o t h on t h e same s i d e o f t h e p y r a n o s e r i n g and t h e d i f f e r e n t s t e r e o - o r i e n t a t i o n o f t h e two h y d r o x y l groups c a n be o b t a i n e d b y 1 4


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F i g . 2 Mechanisms p r o p o s e d f o r t h e p o l y m e r i z a t i o n o f g l u c a n s by g l u c a n s u c r a s e s by a d d i t i o n o f g l u c o s e t o t h e r e d u c i n g end o f t h e glucan chain. (A)

S y n t h e s i s o f d e x t r a n by B-512F

(B)

S y n t h e s i s o f mutan b y mutansucrase.

dextransucrase.

(C)

S y n t h e s i s o f a l t e r n a n by a l t e r n a n s u c r a s e .

Where O ^ r e p r e s e n t s s u c r o s e w i t h Ο b e i n g g l u c o p y r a n o s e and ^ being f r u c t o f u r a n o s e ; O i l represents glucopyranose residue a t t a c h e d t o g l u c o s e r e s i d u e by an a-1->6 l i n k a g e ; Q O r e p r e s e n t s glucopyranose r e s i d u e a t t a c h e d t o g l u c o s e r e s i d u e b y an α-1-+3 l i n k a g e ; X" r e p r e s e n t s a n u c l e o p h i l e a t t h e a c t i v e - s i t e t h a t when a t t a c h e d t o g l u c o p y r a n o s e o r i e n t s t h e g l u c o p y r a n o s e r e s i d u e so t h a t i t s C-6-0H i s i n p o s i t i o n t o a t t a c k C - l o f t h e o t h e r g l u c o p y r a n o s y l r e s i d u e t o form an a-l->6 l i n k a g e ; Y " a l s o r e p r e s e n t s a n u c l e o p h i l e a t t h e a c t i v e - s i t e t h a t when a t t a c h e d t o g l u c o p y r a n o s e o r i e n t s t h e g l u c o p y r a n o s e r e s i d u e so t h a t i t s C-3-0H i s i n p o s i t i o n t o a t t a c k C - l o f t h e o t h e r g l u c o p y r a n o s y l r e s i d u e t o form an 3 b r a n c h l i n k a g e . F i g . 3A shows t h e mechanism f o r t h e s y n t h e s i s o f b r a n c h l i n k a g e s by t h e acceptor r e a c t i o n o f a glucan chain. Many o t h e r a c c e p t o r r e a c t i o n s o c c u r when low m o l e c u l a r w e i g h t c a r b o h y d r a t e s a r e added t o t h e r e a c t i o n d i g e s t . These a c c e p t o r s react with d i f f e r e n t e f f i c i e n c i e s to give d i f f e r e n t kinds o f products (24). The b e s t known a c c e p t o r i s m a l t o s e , f o l l o w e d b y i s o m a l t o s e , n i g e r o s e , a - m e t h y l - D - g l u c o p y r a n o s i d e , D-glucose, l a c t o s e , c e l l o b i o s e i n decreasing order o f t h e i r e f f i c i e n c i e s . Table I gives the r e l a t i v e e f f i c i e n c i e s o f 17 a c c e p t o r s . F o r many o f t h e s e a c c e p t o r s , g l u c o s e i s t r a n s f e r r e d from t h e enzyme t o t h e C-6-0H group a t t h e nonreducing-end t o form a p r o d u c t t h a t i s a l s o an a c c e p t o r . This r e s u l t s i n a homologous s e r i e s o f a c c e p t o r p r o d u c t s i n w h i c h i s o m a l t o s y l c h a i n s o f v a r y i n g l e n g t h a r e a t t a c h e d t o C-6 o f t h e non reducing-end o f the acceptor (13). Other a c c e p t o r s , such as D - f r u c t o p y r a n o s e , l a c t o s e , m e l i b i o s e , D-mannopyranose, and D - g a l a c t o f u r a n o s e , o n l y a s i n g l e p r o d u c t i s formed. The r e s u l t i n g p r o d u c t s have u n u s u a l s t r u c t u r e s . D - f r u c t o pyranose g i v e s the d i s a c c h a r i d e , l e u c r o s e , i n which the glucose i s t r a n s f e r r e d t o C-5 ( 1 5 ) . L a c t o s e g i v e s a t r i s a c c h a r i d e i n w h i c h t h e g l u c o s e i s t r a n s f e r r e d t o C-2 o f t h e r e d u c i n g g l u c o s e r e s i d u e ( 1 6 ) . T h i s i s i n c o n t r a s t t o t h e isomer o f l a c t o s e , c e l l o b i o s e , w h i c h a l s o has t h e g l u c o s e t r a n s f e r r e d t o C-2 o f t h e r e d u c i n g g l u c o s e r e s i d u e ( 1 7 ) , b u t t h i s t r a n s f e r r e d g l u c o s e r e s i d u e c a n a c t as an a c c e p t o r t o g i v e a homologous s e r i e s w i t h i s o m a l t o s y l c h a i n s a t t a c h e d t o C-2 ( 1 4 ) . Thus, t h e i n v e r s i o n o f t h e h y d r o x y l group a t C-4 on t h e non reducing-end glucose residue o f c e l l o b i o s e to give l a c t o s e a f f e c t s the b i n d i n g o f l a c t o s e so t h a t i t s a c c e p t o r p r o d u c t cannot a c t as an a c c e p t o r t o g i v e a homologous s e r i e s as c a n c e l l o b i o s e . T h i s same k i n d o f b i n d i n g impediment i s a l s o o b s e r v e d f o r m e l i b i o s e and r a f f i n o s e b o t h o f which c o n t a i n D - g a l a c t o s e as p a r t o f t h e i r structure. The cause o f t h i s b i n d i n g s p e c i f i c i t y a w a i t s f u r t h e r s t u d y and i n t e r p r e t a t i o n . D-mannose and D - g a l a c t o s e a l s o g i v e u n u s u a l a c c e p t o r p r o d u c t s b o t h o f w h i c h a r e n o n r e d u c i n g sugars (18). D-mannose g i v e s a-Dglucopyranosyl-l->l-j0-D-mannopyranoside, an isomer o f a , 0 - t r e h a l o s e and D - g a l a c t o s e g i v e s o- D- g l u c o p y r a n o s y l -1-*1 -0-D- g a l a c t o f u r a n o s i d e , an isomer o f s u c r o s e ( s e e F i g . 4 f o r t h e s e s t r u c t u r e s ) . The mechanism f o r t h e a c c e p t o r r e a c t i o n s i n v o l v e t h e b i n d i n g o f the a c c e p t o r a t t h e a c t i v e - s i t e i n such a manner t h a t one o f t h e i r h y d r o x y l groups i s s p e c i f i c a l l y o r i e n t e d t o make a n u c l e o p h i l i c a t t a c k onto C - l o f t h e enzyme l i n k e d g l u c o s y l u n i t o r d e x t r a n o s y l


400


r-X© Ι_χΘ

B

Le Fig.

Lxe

3

Mechanisms f o r the s y n t h e s i s o f b r a n c h l i n k a g e s f o r a c c e p t o r r e a c t i o n s o f B-512F d e x t r a n s u c r a s e .

(A)

Synthesis reactions

(B)

Acceptor

o f a-l-+3 b r a n c h linkages o f exogenous d e x t r a n .

by

acceptor

r e a c t i o n s o f maltose

Symbols a r e t h e same as t h o s e

and

d e f i n e d f o r F i g u r e s 1 and 2.


30.

ROBYT

Table I

Quantitative Effects of Acceptors i n the B-512F Dextransucrase Reaction

X Glucose incorporated into acceptors

Acceptor

401


% Glucose incorporated into dextran

No. of acceptor products

.Relative acceptor efficiency 3

Maltose

80.,7

19.,3

5

100.0

Isomaltose

71.,9

28.,1

6

88.7

Nigerose

47.,4

52.,6

6

58.1

a-Me-D-Glc

42.,3

57.J

7

51.5

1,5-anhydrο • D-glueitoi

24..7

75.,3

7

29.9

D-glucose

14..2

85..8

7

17.1

Turanose

11.,2

88..8

7

13.4

/3-Me-D-Glc

10..2

89..8

4

12.3

Lactose

9..0

91,.0

1

10.7

Cellobiose

7..5

92..5

4

9.0

D-Fructose

5,.3

94,.7

1

6.4

Raffinose

3,.7

96 .3

1

4.4

Melibiose

3 .5

96 .5

1

4.2

L-sorbose

2 .6

97 .4

4

3.2

D-Mannose

2 .4

97 .6

1

2.9

D-Galactose

1 .4

98 .6

1

1.7

D-Xylose

0 .4

99 .6

1

0.5

a

R e l a t i v e e f f i c i e n c y d e t e r m i n e d b y comparing t h e amount o f d e x t r a n synthesized i n the p r e s e n c e o f each a c c e p t o r t o t h e amount s y n t h e s i z e d i n t h e p r e s e n c e o f m a l t o s e and a s s i g n i n g 100% e f f i c i e n c y to maltose.



402

Acceptor

Panose Maltose

Isomal totr i ose

Isomaltose

Fructopyranose

Galactofuranose

Mannose

Analogue

Leucrose

α-Glc-p

o-GIc

/?-Gal-f

β-Man

Sucrose

α,/3-Trehalose

a-GIc-(1 — 2 ) - L a c t o s e

Lactose Fig. 4

S t r u c t u r e s o f some a c c e p t o r p r o d u c t s o f B-512F d e x t r a n sucrase.


30. ROBYT


403

chain. T h i s r e s u l t s i n t h e r e l e a s e o f g l u c o s e and/or d e x t r a n from the a c t i v e - s i t e and the f o r m a t i o n o f an α-glucosidic l i n k a g e between t h e a c c e p t o r and g l u c o s e o r d e x t r a n ( 1 3 ) . See F i g . 3B f o r the mechanism o f t h e a c c e p t o r r e a c t i o n . α-Methyl-D-glucopyranoside i s a more e f f i c i e n t a c c e p t o r t h a n Dg l u c o s e , which i n t u r n i s a b e t t e r a c c e p t o r than -methyl-D-gluco pyranoside (14). The acceptor binding s p e c i f i c i t y f o r mono saccharides has been studied using α-methyl-D-glucopyranoside a n a l o g u e s , m o d i f i e d a t C-2, C-3, and C-4 p o s i t i o n s by (a) i n v e r s i o n o f the h y d r o x y l groups and (b) r e p l a c e m e n t o f the h y d r o x y l group w i t h h y d r o g e n ( 1 9 ) . The r e l a t i v e e f f e c t i v e n e s s o f each o f the a n a l o g u e s i s g i v e n i n T a b l e I I a l o n g w i t h the p o s i t i o n o f a t t a c h m e n t o f the g l u c o s e r e s i d u e i n the a c c e p t o r p r o d u c t . D-Glucopyranose i s t r a n s f e r r e d t o the C-6-0H o f the analogues so m o d i f i e d a t C-2 and C-3; i t i s t r a n s f e r r e d t o C-4-0H o f the 4 - i n v e r t e d analogue, and t o t h e C-3OH o f the 4-deoxy-analogue. The r e l a t i v e e f f i c i e n c y o f the a c c e p t o r r e a c t i o n s w i t h t h e s e analogues i n d i c a t e s t h a t t h e h y d r o x y l group a t C-2 i s n o t as i m p o r t a n t f o r a c c e p t o r b i n d i n g as the h y d r o x y l groups a t C-3 and C-4. The h y d r o x y l group a t C-4 i s p a r t i c u l a r l y i m p o r t a n t as i t d e t e r m i n e s the b i n d i n g o r i e n t a t i o n o f the a c c e p t o r p y r a n o s e ring. An i n t e r e s t i n g r e s u l t i s o b t a i n e d f o r C-6 m o d i f i e d a n a l o g u e s , α - m e t h y l - 6 - d e o x y - D - g l u c o p y r a n o s i d e and α-methyl-6-deoxy-6-fluorο-Dg l u c o p y r a n o s i d e , n e i t h e r o f which are e i t h e r a c c e p t o r s or i n h i b i t o r s o f d e x t r a n s y n t h e s i s . I t i s c o n c l u d e d t h a t n e i t h e r a r e bound a t a l l (19). T h i s o b s e r v a t i o n i n d i c a t e s t h a t the a c c e p t o r b i n d i n g s i t e i s d i s t i n c t from the s u c r o s e b i n d i n g s i t e as b o t h 6-deoxy- and 6 - f l u o r o s u c r o s e a r e p o t e n t c o m p e t i t i v e i n h i b i t o r s f o r d e x t r a n s y n t h e s i s (20) . I f the 6 - m o d i f i e d α-methyl-D-glucopyranos i d e s were bound a t t h e s u c r o s e s i t e , i t would be e x p e c t e d t h a t the 6-deoxy- and 6 - f l u o r o analogues of α-methyl-D-glucopyranoside would also be potent inhibitors. Water a l s o i s an a c c e p t o r g i v i n g a p p r o x i m a t e l y 7% h y d r o l y s i s o f sucrose (14). The f i r s t a c c e p t o r p r o d u c t f o r the r e a c t i o n w i t h m a l t o s e i s the t r i s a c c h a r i d e , panose ( 6 - a - g l u c o p y r a n o s y l m a l t o s e ) (13,21). The amount o f d e x t r a n s y n t h e s i z e d d e c r e a s e s e x p o n e n t i a l l y as the r a t i o o f m a l t o s e t o s u c r o s e i n c r e a s e s (14) and the number o f homologous p r o d u c t s a l s o d e c r e a s e (Su, Ό.Ί.; Fu, D., and Robyt, J.F., unpublished results). The use of relatively high c o n c e n t r a t i o n s o f m a l t o s e i n the r e a c t i o n d i g e s t has b e e n u s e d as a p r e p a r a t i v e method f o r panose (21). Similarly, high concentrations of D- f r u c t o s e i n the r e a c t i o n d i g e s t has been u s e d t o p r e p a r e l e u c r o s e (15). The s t r u c t u r e s o f the p r o d u c t s r e s u l t i n g from the r e a c t i o n o f the h i g h e r m a l t o d e x t r i n s , G3--G8, have b e e n d e t e r m i n e d (Fu, D.; Robyt, J.F., unpublished results). Each o f t h e s e m a l t o d e x t r i n s gave two p r o d u c t s : the f i r s t , and major p r o d u c t , had the g l u c o s e a t t a c h e d to C-6 o f the n o n r e d u c i n g g l u c o s e r e s i d u e and t h e second, had the g l u c o s e a t t a c h e d t o C-6 o f the r e d u c i n g g l u c o s e r e s i d u e . The former p r o d u c t s were a l s o a c c e p t o r s t o g i v e a homologous s e r i e s w i t h isomaltosyl chains of various sizes attached t o C-6 of the n o n r e d u c i n g g l u c o s e r e s i d u e o f the m a l t o d e x t r i n s , w h i l e the a c c e p t o r 2



a

81.7 92.5

18,.2 16,.9 6. 2 4.,0 2.,9 0.,0 0. 0 0.,0

a-Me-Man-p

3-deoxy-a-Me-Glc-p

a-Me-All-p

a-Me-Gal-p

4-deoxy-a-Me-Glc-p

6-deoxy-a-Me-Glc-p

6-fluoro-a-Me-Glc-ρ

a-Me-Xyl-p

55.4

32 .0

0.0

-

-

-

0.0

0.0

6.6

9.2

14.2

39.0

42.0

73.7

100.0

8

Relative efficiency

2

1

5

8

6

7

10

No. of Acceptor product

C-3

C-4

C-6

C-6

C-6

C-6

C-6

Position of linkage

R e l a t i v e e f f i c i e n c y d e t e r m i n e d by comparing t h e amount o f d e x t r a n s y n t h e s i z e d i n the p r e s e n c e o f each a n a l o g u e t o the amount s y n t h e s i z e d i n t h e p r e s e n c e o f α-methyl-D-glucopyranoside and a s s i g n i n g 100% e f f i c i e n c y t o a - m e t h y l - D - g l u c o pyranoside.

100.0

100.0

100.0

95.5

94.2

80.4

67.3

43 .4

2-deoxy-a-Me-Glc-p

% Glucose incorporated into dextran

a-Me-Glc-p

Acceptor

X Glucose incorporated into acceptor

Table I I Quantitative E f f e c t s o f α-Methyl-D-glucopyranoside Analogues as Acceptors i n the B-512F Dextransucrase Reaction

30. ROBYT

405


product with glucose attached to C-6 of the reducing glucose residue were very poor acceptors, i f they were acceptors at a l l . The e f f i c i e n c y of the maltodextrins as acceptors decreases as the chain length increases. With the efficiency of maltose being assigned 100%, the efficiency of maltotriose drops to 40%, and maltooctaose to

only 6% (Fu, D.; Robyt,

J.F. , unpublished

Going

results).

to

larger maltodextrin chains, i t would be expected that the e f f i c i e n c y i s even lower. Attempts to form acceptor products with starch have been unsuccessful. The maltodextrin acceptors are unusual i n that both the nonreducing glucose residue and the reducing glucose residue are s i t e s for glucose transfer from the enzyme. This means that an asymmetric maltodextrin molecule can be bound i n the acceptor s i t e at either end and that the binding of the maltodextrin i s i n such a manner that i t i s not allowed to "slide" along the binding s i t e and accept the transfer of glucose at any of the i n t e r i o r glucose residues. This is i n contrast to dextran chains that can accept glucose and glucan at i n t e r i o r glucose residues to give a branched dextran product (11). The reaction of maltose as an acceptor with alternansucrase gave panose as the f i r s t product. There were two acceptor products formed from panose: (a) the transfer of glucose to C-6 of the nonreducing residue to give 6 -a-isomaltosyl maltose and (b) the transfer of glucose to C-3 of the nonreducing residue to give 6 -a-nigerosyl maltose. The l a t t e r product i s an unusual structure that has a sequence of glycosidic linkages from the reducing-end of (α-1-*4) , (a1-+6), and (a-l-»3) (22). Isomaltose also gave two products i n which (a) glucose was transferred to C-6 of the nonreducing-end to give isomaltotriose and (b) glucose was transferred to C-3 of the nonreducing-end to give 3 -a-glucopyranosyl isomaltose. The s p e c i f i c i t y of alternansucrase for acceptor reactions, thus, requires f i r s t the formation of an a-1->6 linkage before the formation of an a-l-+3 l i n k age, but the presence of an α-1-*·6 does not preclude the formation of an a-1->6 linkage. Recently, i t has been reported that a low molecular weight alternan, with lowered v i s c o s i t y , can be produced by conducting the synthesis of alternan i n the presence of D-glucose and endodextranase (23). The l a t t e r prevents the formation of dextran by contaminating dextransucrase and the former decreases the molecular size of the alternan by acceptor reactions that terminates alternan poly merization. B-512F Dextransucrase has also been used i n conjunction with endodextranase to produce a product with a high content of isomaltose and D-fructose (24). The D-fructose can be removed by a Ca-cation exchange r e s i n , giving an isomaltose syrup. The isomaltose can then be c a t a l y t i c a l l y reduced to i s o m a l t i t o l , which i s sweet and of low c a l o r i c value. In this paper, i t has been shown that glucansucrases with different synthetic s p e c i f i c i t i e s can synthesize glucans with different structures and can synthesize oligosaccharides with different structures by acceptor reactions. The mechanisms by which these enzymes carry out the polymerization and by which the acceptor reactions occur has been discussed. The products r e s u l t i n g from the 2

2

2


406


action of these enzymes are potentially new products that could be produced from the biomass material, sucrose, and could have new uses and applications. Literature Cited 1.

Jeanes, Α.; Haynes, W. C.; William, C. Α.; Rankin, J. C.; Melvin, Ε. H.; Austin, M. J.; Cluskey, J. E.; Fisher, Β. E.; Tuschiya, H. M.; Rist, C. Ε., J. Amer. Chem. Soc. 1954,76,5041-4. 2. Wilham, C. Α.; Alexaqnder, Β. H.; Jeanes, Α., Arch. Biochem. Biophys. 1955,59,61-6. 3. Robyt, J. F., "Dextran", in Encyclopedia of Polymer Science and Engineering, H. F. Mark, N. M. Bikales, C. G. Overberger, and G. Menges, Eds. 2nd Ed.; John Wiley & Sons, NY 1986, Vol. 4; 752-767. 4. Shimamura, Α.; Tsumori, H.; Mukasa, Η., Biochim. Biophys. Acta 1982,702,72-8. 5. Robyt, J. F.; Martin, P. J., Carbohydr. Res. 1983,113,301-15. 6. Cote, G. L, ; Robyt, J. F., Carbohydr. Res. 1982,101,57-74. 7. Robyt, J. F.; Walseth, T. F., Carbohydr. Res. 1979,68,95-111. 8. Miller, A. W.; Eklund, S. H.; Robyt, J. F., Carbohydr. Res. 1986,147,119-33. 9. Fu, D.; Robyt, J. F., Prep. Biochem. in press 1990. 10. Robyt, J. F.; Kimble, Β. K.; Walseth, T. F., Arch. Biochem. Biophys. 1974,165,634-40. 11. Robyt, J. F.; Taniguchi, Η., Arch. Biochem. Biophys. 1976,174,129-34. 12. Robyt, J. F.; Corrigan, A. J., Arch. Biochem. Biophys. 1977,183,726-31. 13. Robyt, J. F.; Walseth, T. F., Carbohydr. Res. 1978,61,433-45. 14. Robyt, J. F.; Eklund, S. Η., Carbohydr. Res. 1983,121,279-86. 15. Stodola, F. H.; Sharpe, E. S.; Koepsell, H. J., J. Amer. Chem. Soc. 1956,78,2541-8. 16. Bourne, E. J.; Harrington, J.; Weigel, Η., J. Chem. Soc. 1959,2332-6. 17. Bourne, E. J.; Harrington, J.; Weigel, Η., J. Chem. Soc. 1961,1088-92. 18. Iriki, Y.; Hehre, E. J., Arch. Biochem. Biophys. 1969,134,130-6. 19. Fu, D.; Slodki, M. E.; Robyt, J. F., Arch. Biochem. Biophys. 1990,276,460-5. 20. Eklund, S. H.; Robyt, J. F., Carbohydr. Res. 1988,177,253-8. 21. Killey, M.; Dimler, R. J.; Cluskey, J. Ε., J. Amer. Chem. Soc. 1955,77,3315-8. 22. Cote, G. L.; Robyt, J. F., Carbohydr. Res. 1982,111,127-42. 23. Cote, G. L., Abst. Papers, 199th Amer. Chem. Soc. Meetin 1990, No. 49 CARB, Boston, MA. 24. Paul, F. B.; Monsan, P. F; Remand, M. M. C.; Pelenc, V. P., U.S. Patent No. 4,861,381,1989. RECEIVED August 16, 1990


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