Biotechnology of Amylodextrin Oligosaccharides - American Chemical

Chapter 8

Strategies for the Specific Labeling of Amylodextrins Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch008

John F. Robyt Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011

Maltodextrins and isomaltodextrins specifically labeled in (a) the reducing-end glucopyranosyl unit, (b) the nonreducing-end glucopyranosyl unit, (c) a specific number of labeled glucopyranosyl units at one or the other end or both of the chain ends, and (d) a l l of the glucopyranosyl units uniformly labeled, find uses in the study of the function of the dextrins and especially in the study of the mechanisms of enzymes that interact with the dextrins and related substrates. Branched maltodextrins and cyclodextrins can also be specifically labeled. A l l of the methods that w i l l be discussed involve the use of specific enzymes that are commer c i a l l y available or can be readily prepared in the laboratory.

Synthesis o f uniformly labeled Maltodextrins C-14-Uniformly labeled glycogen can be r e a d i l y synthesized using uniformly labeled C-14-sucrose and Neisseria perflava amylosucrase (1,2). The labeled glycogen can be converted into l i n e a r a-l->4 linked maltodextrins by the action of Pseudomonas amyloderma isoamylase that s p e c i f i c a l l y hydrolyzes the a-1->6 branch linkages of glycogen to give a mixture of uniformly labeled maltodextrins of degree of polymerization (D.P) 10 and greater. These can be converted into s p e c i f i c maltodextrins of D.P. 2 to 6 by the action of s p e c i f i c amylases. For example, maltose can be obtained from the action of ^-amylases; maltotriose from the action of G3-amylase from Streptomyces griseus (3); maltotetraose from the action of G4-amylase from Pseudomonas stutzeri (4); maltopentaose from the action of G5-amylase from Pseudomonas sp. (5); and maltohexaose from the action of G6-amylase from Aerobacter aerogenes (6) . A mixture of maltose — maltoheptaose can be obtained by the action of B. amyloliquefaciens α - a m y l a s e i n which d i f f e r e n t amounts of 0097-6156/91/0458-0098$06.00/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch008

8.

ROBYT

Strategies for the Specific Labeling of Amylodextrins 99

maltodextrins can be obtained, depending on the amount of enzyme used and the length of time of the reaction (7). Uniformly labeled cyclodextrins can be synthesized by the action of B. macerans cyclodextrin glucanosyl transferase acting on C-14 labeled amylo dextrins obtained from the action of N. perflava amylosucrase and C-14 sucrose. See Table Γ for a l i s t of sources of the enzymes. Recently, we synthesized maltotetraose s p e c i f i c a l l y labeled with C-13 at carbon-1 of each glucopyranosyl residue by converting 1-C-13-enriched D-glucose into 1-C-13-a-glucopyranosyl f l u o r i d e , which i s a substrate for N. perflava amylosucrases to give 1-C-13glycogen. The 1-C-13-glycogen was reacted with isoamylase to cleave the a-l-+6 branch linkages, followed by reaction with P. stutzeri G4 amylase to give the l-C-13-labeled maltotetraose (8) (Figure 1). This can be considered a special type of uniformly labeled maltodextrin i n which each of the glucopyranosyl residues are labeled at C - l with C-13. Synthesis of Uniformly Labeled Isomaltodextrins A series of C-14-uniformly labeled isomaltodextrins can be obtained by the action of Leuconostoc mesenteroides B-512F dextransucrase and U-C-14-sucrose and U-C-14-glucose. The glucose acts as an acceptor to which dextransucrase catalyzes the transfer of the glucopyranosyl moiety of sucrose to the C-6-0H of the glucose acceptor (9,13); this product, isomaltose can then act as an acceptor to give isomaltot r i o s e , which i n turn i s an acceptor to give isomaltotetrose, isomaltopentaose, etc. The number and amount of each isomaltodextrin dependent on the r e l a t i v e concentration r a t i o of sucrose and glucose (9). Using an equimolar amount of sucrose and glucose gives isomaltodextrins down to D.P. 7. The amounts of the isomal todextrins, however, decrease as the size of the dextrins become larger (9). Synthesis of Reducing-end Labeled Maltodextrins Reducing-end labeled maltodextrins can be prepared by the acceptor or coupling reaction catalyzed by B. macerans cyclodextrin glu canosyl transferase reaction between nonlabeled cyclomaltohexaose with C-14 D-glucose (10). The f i r s t labeled product i s maltoheptaose, which i s s p e c i f i c a l l y labeled i n the reducing-end glucopyranosyl u n i t . This reducing-end labeled maltoheptaose then undergoes a series of disproportionation reactions i n which i n i t i a l l y two maltoheptaose molecules react. For example, one of the disproportionation reactions could give maltose and maltododecaose, a second could give maltotriose and maltoundecaose, etc. The products of the disproportionation reactions of maltoheptaose themselves can undergo disproportionation reactions (Figure 2). A l l of these reactions give a homologous series of maltodextrins, s p e c i f i c a l l y and exclusively labeled i n the reducing-end gluco pyranosyl residue (20). Several other acceptors other than D-glucose could also be used, e . g . , maltose, sucrose, a-methyl-Dglucopyranoside, isomaltose, and maltooligosaccharides to give homologous series i n which the acceptor i s located at the reducing-



100

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

Table I

Neissera

Sources o f Enzymes Used to Prepare S p e c i f i c a l l y Labeled Amylodextrins

perflava

amylosucrase

Prepared from culture,

réf. 1

Bacillus amyloliquefaciens α-amylase

Sigma Chemical C o . , St. Louis, MO

Streptomyces griseus

G3-amylase


ref.

3

Pseudomonas stutzeri

G4-amylase


ref.

4

G5-amylase


ref.

24

aerogenes G6-amylase

Prepared from c u l t u r e ,

ref.

6

Pseudomonas sp. Aerobacter

Pseudomonas amyloderma isoamylase

Sigma Chemical Company

B. macerans cyclodextrin glucanosyl transferase

Amano International Enzyme Company, I n c . , Troy, VA

Leuconostoc mesenteroides dextransucras e

B-512F


Pullulanase

Amano International Company, Inc.

Enzyme

Muscle phosphorylase


β-amylase




8. ROBYT

13


C¥H,OH

a-D-glucopyranosyl fluoride amylosucrase

>•

13 1- O G L Y C O G E N

(1) i s o a m y l a s e (2) P. s t u t z e r i 13

C-Malto tetraose

amylase

Figure 1. Synthesis of 1-C-13-maltotetraose, using amylosucrase, isoamylase, and Pseudomonas stutzeri G4-amylase.



^

transferase

r e a c t i o n s

-

0

G,

0

OOOOO0

0

e«

0

G

(ΚΗ>0< 0

^

ChO-p

o-#

Figure 2. Synthesis of reducing-end labeled maltodextrins by reaction of Bacillus macerans cyclodextrin glucanosyl transferase with cyclomaltohexaose and U-C-14-D-glucose. Reaction takes place in two steps: (A) the coupling reaction to give reducing-end labeled maltoheptaose and (B) disproportionation reactions, first between two maltoheptaose molecules and then between the various resulting disproportionation products. The circle represents a glucopyranosyl unit and a circle with a slash represents a reducing glucopyranose unit; black are labeled and white are unlabeled.

O-0H3-Q-O-O-0

ooo-oo-o-#

^

CH>CK>CK>-# «»

disproportionation

Cyclodextrin glucanosyl

+ # -—:

M


8. ROBYT


end or the potential reducing-end of chain (11) (Figure 3).

the

r e s u l t i n g maltodextrin


Synthesis of Reducing-end Labeled Isomaltodextrins C-14 Reducing-end labeled isomaltodextrins can be synthesized by using nonlabeled sucrose and C-14 labeled D-glucose or C-14 labeled α - m e t h y l - D - g l u c o p y r a n o s i d e with L . mesenteroides B-512F dextran sucrase (9,12,13). The labeled acceptor w i l l be s p e c i f i c a l l y located at the reducing or potential reducing-end (13) (Figure 4). As with the B. macerans cyclodextrin glucanosyl transferase, several d i f f e r e n t acceptors can be used. Over t h i r t y d i f f e r e n t acceptors have been i d e n t i f i e d (14). Not a l l of them, however, react with equal e f f i c i e n c y . The best acceptor i s maltose, followed by isomaltose, nigerose, α - m e t h y l - D - g l u c o p y r a n o s i d e , 1,5-anhydro-Dg l u c i t o l , and D-glucose with e f f i c i e n c i e s r e l a t i v e to maltose of 89, 58, 52, 30, and 17 percent, respectively (9). When the r a t i o of maltose to sucrose i s r e l a t i v e high i n the dextransucrase acceptor reaction, the branched t r i s a c c h a r i d e , panose, i s the major product (12) (Figure 5). Thus, by using U-C-14 sucrose and nonlabeled maltose, nonreducing-end labeled panose i s formed. Using d i f f e r e n t types of acceptors, either labeled or nonlabeled, d i f f e r e n t series of isomaltodextrins are produced with the d i f f e r e n t kinds of acceptors located at the reducing end of the chains. Synthesis of Labeled Branched Maltodextrins When other maltodextrins, such as maltotriose, are used as acceptors i n the dextransucrase-sucrose reaction, two acceptor products are formed i n which the glucopyranosyl moiety of sucrose i s transferred to the C-6-0H group of the nonreducing-end glucose residue and to the reducing glucose residue of maltotriose. This gives 6 -a-Dglucopyranosyl maltotriose and 6 -a-D-glucopyranosyl maltotriose (15) (Figure 5). The former has the structure of the smallest α - a m y l a s e l i m i t dextrin (B4) formed i n the hydrolysis of amylopectin by most α - a m y l a s e s (16,17). Thus, when the maltotriose i s nonlabeled and the sucrose i s labeled, a nonreducing-end labeled B4 i s synthesized. An interesting double-labeled saccharide would r e s u l t with l a b e l i n both the reducing-end and the nonreducing-end when both reducing-end labeled maltotriose and labeled sucrose are used (reaction Β of Figure 6). Similar kinds of reactions w i l l occur when maltotetraose i s the acceptor to give 6*-a-D-glucopyranosyl maltotetraose and 6 -a-D-glucopyranosyl maltotetraose (reaction C of Figure 5). 3

1

1

Synthesis of Nonreducing-end Labeled Maltodextrins Nonreducing-end labeled maltodextrins of D.P. 5 and larger can be synthesized using the reaction of nonlabeled maltotetraose acceptor with labeled a-D-glucopyranosyl-1-phosphate, catalyzed by muscle phosphorylase, which w i l l give nonreducing-end labeled maltopentaose (Figure 7). Maltotetraose is the smallest acceptor that can be used and hence, the smallest possible nonreducing-end labeled malto dextrin is maltopentaose. Maltoheptaose was used as an acceptor



— CKM>CK>-0-#-^

-

Μβ

1

tose

Sucrose

Isomaltose

Mal

α-Methy l - D -

F i g u r e 3. Synthesis o f reducing-end l a b e l e d m a l t o d e x t r i n s by r e a c t i o n o f Β. macerans c y c l o d e x t r i n g l u c a n o s y l t r a n s f e r a s e w i t h c y c l o m a l t o h e x a o s e and v a r i o u s k i n d s o f a c c e p t o r s . Symbols a r e t h e same as i n F i g u r e 2.

ο

Q-CK>-CK>C>-#^

— 0--·^

φ-OMe "—

#

-— C K X X X X H *

at

reducing-end

Residues


the


8.

ROBYT


F i g u r e 4. S y n t h e s i s o f (A) r e d u c i n g - e n d l a b e l e d isomaltod e x t r i n s by r e a c t i o n o f dextransucrase w i t h n o n l a b e l e d sucrose and l a b e l e d s u c r o s e and l a b e l e d a-methyl-D-glucopyranoside; s y n t h e s i s o f (B) n o n r e d u c i n g - e n d i s o m a l t o d e x t r i n s b y r e a c t i o n w i t h l a b e l e d s u c r o s e and n o n l a b e l e d i s o m a l t o s e ; and s y n t h e s i s o f (C) d u a l l a b e l e d i s o m a l t o d e x t r i n s b y r e a c t i o n w i t h l a b e l e d sucrose and r e d u c i n g - e n d labeled isomaltose. The c i r c l e r e p r e s e n t s a g l u c o p y r a n o s y l u n i t and a c i r c l e w i t h a s l a s h r e p r e s e n t s a reducing glucopyranose u n i t ; a t r i a n g l e r e p r e s e n t s t h e f r u c t o s e u n i t ; b l a c k a r e l a b e l e d and w h i t e a r e u n l a b e l e d .



106


+ O-0

—**~J-0

Β

+ O-O-O-0

— * & - O O 0

+

ο-ο-ο^ό

Figure 5. Sjnithesis of nonreducing-end labeled branched malto dextrins by reaction of dextransucrase with labeled sucrose and nonlabeled maltodextrins: reaction with (A) maltose, (B) maltotriose, and (C) maltotetraose. Symbols are the same as i n Figure 4.

α

2κ># + o-i

Figure 6. Synthesis of variously labeled branched maltotetrasaccharides by reaction of dextransucrase with (A) nonlabeled sucrose and reducing-end labeled maltotriose, (B) labeled sucrose and reducing-end labeled maltotriose, and (C) labeled sucrose and nonlabeled maltotriose. Symbols are the same as i n Figure 4.



^ Ρ,·

^

# - 0 0 0 0 Nonreducing-end Labeled M a l t o p e n t a o s e

P:

"ν-—-t-OOCHl

phosphory la se

—

Figure 7. Synthesis of the smallest nonreducing-end labeled maltodextrin, maltopentaose, (A) by r e a c t i o n of phosphorylase with nonlabeled maltotetraose and labeled a-glucopyranosyl1-phosphate; and dual labeled maltopentaose (B) by reaction with reducing-end labeled maltotetraose and labeled a-gluco pyranosyl -1 -phosphate . Symbols are the same as i n Figure 2.

ΟΟΌ-φ

Maltotetraose

G-l-P

φ-ο'

OOO-0

·*'


Β

/

Org

I

108

BIOTECHNOLOGY OF AMYLODÉXTRIN OLIGOSACCHARIDES


to give nonreducing-end labeled maltooctaose (18). When equimolar amounts of maltoheptaose and C-14-a-glucopyranosyl-l-phosphate were used, nonreducing-end labeled maltooctaose was the major product with a small amount of labeled maltononaose, which was labeled i n the l a s t two glucopyranosyl residues at the nonreducing-end. Dual labeled maltodextrins can be formed with the glucose residues labeled at both the nonreducing-end and the reducing-end by reaction of muscle phosphorylase with reducing-end labeled maltodextrin and labeled a-glucopyranosyl-1-phosphate (Figure 7). Synthesis of Nonreducing-end Labeled Isomaltodextrins Nonreducing-end isomaltodextrins can be synthesized by dextransucrase, sucrase, s t a r t i n g with nonlabeled isomaltodextrin acceptor and labeled sucrose. For example, s t a r t i n g with nonlabeled isomaltose and labeled sucrose, nonreducing-end labeled isomaltotriose would be formed, and s t a r t i n g with nonlabeled isomaltotriose and labeled sucrose, nonreducing-end isomaltotetraose would r e s u l t . Dual labeled isomaltodextrins can also be synthesized, s t a r t i n g with reducing-end labeled isomaltodextrin acceptors and labeled sucrose (Figure 4). Synthesis of Labeled Maltosyl Branched-cyclomaltodextrins Labeled maltosyl branched-cyclomaltodextrins can be synthesized by the action of isoamylase (19-20) or pullulanase (22,23) with various types of labeled maltose and cyclodextrin. Three types of labeled maltosyl cyclodextrins can be obtained: (a) reducing-end labeled maltose attached to cyclodextrin; (b) uniformly labeled maltose attached to cyclodextrin; and (c) nonlabeled maltose attached to uniformly labeled cyclodextrin (Figure 8).

We, thus, have shown how a wide v a r i e t y of amylodextrins can be s p e c i f i c a l l y labeled i n different ways by using twelve d i f f e r e n t kinds of enzymes with different kinds of labeled glucosyl donors and different kinds of labeled acceptors or by using combinations of different enzymes i n sequence or by using combinations of labeled glucosyl donors and labeled acceptors together to give dual labeled products. The type of isotope that mostly has been discussed i s C-14, but other types could equally be used, such as C-13 or H-3. As a summary, l e t us l i s t the s p e c i f i c enzymes that can be used i n conjunction with i s o t o p i c a l l y labeled substrates to give s p e c i f i c a l l y labeled amylodextrins: N. perflava amylosucrase; s p e c i f i c exo-acting amylases, such as, 0-amylase, S. griseus G3-amylase, P. stutzeri G4-amylase, Pseudomonas sp. G5-amylase, and A. aerogenes G6-amylase; endo-acting amylases, such as, B. amyloliquefaciens amylase; P. amyloderma isoamylase; B. macerans cyclodextrin glucanosyl-transferase; L . mesenteroides B-512F dextransucrase; pullulanase; and muscle phosphorylase. Sources for these enzymes are given i n Table 1.



8.

ROBYT

Strategies for the Specific Labeling of Âmylodextrins

Pullulanase or Isoamylase F i g u r e 8. S y n t h e s i s o f l a b e l e d b r a n c h e d c y c l o m a l t o h e x a o s e by r e a c t i o n of p u l l u l a n a s e or isoamylase w i t h cyclomaltohexaose and (A) u n i f o r m l y l a b e l e d m a l t o s e , (B) r e d u c i n g - e n d l a b e l e d m a l t o s e , and (C) n o n l a b e l e d m a l t o s e and u n i f o r m l y l a b e l e d cyclomaltohexaose. Symbols a r e the same as i n F i g u r e 2.


109

110


Literature Cited

Okada, G.; Hehre, E. J., J. Biol. Chem. 1974, 249, 126-35. Tao, Β. Y.; Reilly, P. J.; Robyt, J . F., Carbohydr. Res. 1988, 181, 163-74. 3. Wako, K.; Takahashi, E . ; Hashimoto, S.; Kanaeda,J.,Denpun Kagaku 1978,25(1978),155-60. 4. Robyt, J . F.; Ackerman, R. J., Arch. Biochem. Biophys. 1971,145, 105-12. 5. Kobayashi, S.; Okemoto, H.; Hara, K.; Hashimoto Η., Agric. Biol. Chem. 1990, 54, 147-56. 6. Kainuma, K.; Wako, K.; Kobayashi, S.; Nogami, Α.; Suzuki, S. Biochim. Biophys. Acta 1975,410,333-41. 7. Robyt, J . F.; French, D., Arch. Biochem. Biophys. 1963,100,45167. 8. Tao, Β. Y.; Reilly, P. J.; Robyt, J . F., Biochim. Biophys. Acta 1989,995,214-20. 9. Robyt, J . F.; Eklund, S. Η., Carbohydr. Res. 1983,121,279-86. 10. Pazur, J . H., J. Amer. Chem. Soc. 1955,77,1015-8. 11. Norberg, E.; French, D., J. Amer. Chem. Soc. 1950,72,1202-4. 12. Jones, R. W.; Jeanes, Α.; Stringer, C. S.; Tsuchiya, Η. Μ., J . Amer. Chem. Soc. 1956,78,2499-502. 13. Robyt, J . F.; Walseth, T. F., Carbohydr. Res. 1978,61,433-45. 14. Robyt, J . F.; Eklund, S. Η., Bioorganic Chem. 1982,11,115-32. 15. Fu, D.; Robyt, J . F., Arch. Biochem. Biophys. in press 1990. 16. Kainuma, K.; French, D., FEBS Letters 1969,5,257-60. 17. Kainuma, K.; French, D., FEBS Letters 1970,6,182-5. 18. Robyt, J . F.; French, D., J. Biol. Chem. 1970,245,3917-27. 19. Abe, J.; Mizowaki, N.; Hizukuri, S.; Koizumi, K.; Utamura, T., Carbohydr. Res. 1986,154,81-6. 20. Kitahata, S.; Yoshimura, Y.; Okada, S., Carbohydr. Res. 1987,159, 303-8. 21. Hizukuri, S.; Abe, J.; Koizumi, K.; Okada, Y.; Kubota, Y.; Sakai, S.; Mandai, T., Carbohydr. Res. 1989,185,191-8. 22. Abdullah, M.; French, D., Arch. Biochem. Biophys. 1970,137,4835. 23. Shiraishi, T.; Kusano, S.; Tsumuraya, Y.; Sakano, Y. Agric. Biol. Chem. 1989,53,2181-8. 24. Okemoto, H.; Kobayashi, S.; Momma, M.; Hashimoto, H.; Hara, K.; Kainuma, K.; Appl. Microbiol. Biotechnol. 1986,25,137-42. 25. Fu, D.; Robyt, J.F., Prep. Biochem. 1990,20,93-106.


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Biotechnology of Amylodextrin Oligosaccharides - American Chemical

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