Susceptibility of leucrose to carbohydrases

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 70, No. 4, 277-279. 1990

Susceptibility of Leucrose to Carbohydrases M A S A R U I I Z U K A , 1. M A S A T O H I Y A M A , l K U N I K O I T A Y A , 1 K I M I A K I F U R U I C H I , 1 Y O N G - G E U N A N N , l N O S H I M I N A M I U R A , 1 AND T A K E H I K O Y A M A M O T O 2

Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558,1 and Department of Bioengineering, Faculty of Engineering, Fukuyama University, Higashimura, Fukuyama 729-02, 2 Japan Received 22 May 1990/Accepted 20 July 1990 Oligosaccharides were synthesized from sucrose using the dextransucrase o f Leuconostoc mesenteroides, and the products were isolated by chromatography. The oligosaccharide obtained as lhe main product was identified as leucrose by 13C-NMR. P r o d u c t i o n o f leucrose from sucrose was greatly increased when the reaction was d o n e with fructose as an acceptor. The susceptibility o f leucrose to carbohydrases was tested, and leucrose was also a useful substrate for detecting or measuring a-glucosidase in e n z y m e mixtures such as culture filtrates o f microorganisms.

butanol-pyridine-water (8 : 1 : 1) developing three times at 37°C with ethanol-HzSO4 (9 : 1, v/v) as the locator, and by High P e r f o r m a n c e Liquid C h r o m a t o g r a p h y ( H P L C ) using Shimpack SCR 10IN (0.79 × 30 cm) with distilled water as an eluent at a flow rate of 1.0 ml per minute, monitoring with a Knauer differential refractometer. Also, 13C-NMR (Nuclear Magnetic Resonance Spectroscopy) o f (V) was done for identification with a JEOL-GX-400 N M R spectrometer operating at 1 0 0 M H z in D20 at 40°C. Chemical shifts were measured with sodium-4,4-dimethyl-4-sila-pentane sulfonate (DSS) as an internal standard. The p r o d u c t was found to be a disaccharide, with a mobility between maltose and maltotriose, with 1 mol o f glucose and 1 mol o f fructose, judging from TLC spots and H P L C peaks with the sample and hydrolysis products o f the sample by hydrochloric acid (0.1 N HC1, 100°C 12 h). 13C-NMR spectrum of the product (V) was in fair agreement with the d a t a for leucrose (5-O-a-D-glucopyranosyl-D-fructopyranose) reported by Jarrel et al. (13). The susceptibility o f leucrose to carbohydrases was examined by incubating a mixture o f 0.5 mg o f leucrose and 1 unit o f the enzyme in 1.0 ml o f 0.05 M acetate buffer, p H 5 . 2 , at 37°C, except the reaction with yeast aglucosidase, which was done at 30°C. The enzymes used were Bacillus subtilis a-amylase (BSA and BLA) and Rhizopus niveus glucoamylase from U e d a Chemical Ind. Co., Ltd., Osaka, yeast a-glucosidase from T o y o b o Co., Ltd., Osaka, bovine and porcine aglucosidases that were prepared by the method of Hibi et al. (14), h u m a n urinary a-glucosidase (F-I and F-III) (15), Candida utilis fl-fructofuranosidase (16), and Chaetomium gracile dextranase, which was a kind gift o f Dr. A. H a t t o r i of Sankyo Co., Ltd., (17). One unit o f enzyme activity was defined as the a m o u n t o f enzyme that liberates l ~ m o l o f reducing sugar as glucose from maltotriitol per minute for a-glucosidase and 1/lmol o f fructose from sucrose per minute for fl-fructofuranosidase. F o r glucoamylase and a-amylase, one unit o f enzyme was defined as the a m o u n t o f enzyme that produces 1/tmol o f reducing sugar equivalent to glucose from soluble starch (1% solution) under the optimal conditions. The reaction products were measured by the m e t h o d of Somogyi and Nelson and by H P L C . Only ~-glucosidases can act on leucrose to produce glucose and fructose, but

Dextransucrase (EC 2.4.1.5) is the enzyme that transfers a glucose unit from sucrose to another sucrose molecule to form an a-l,6-glucosidic linkage, although some oligosaccharides are formed simultaneously as by-products. M a n y works have been reported so far on dextransucrase (1-5) and on the synthesis o f oligosaccharides (610), but studies on the usage of the resulting oligosaccharides are not numerous. We tried to synthesize some oligosaccharides with acceptor sugars using dextransucrase and tried to test the susceptibility o f the resulting oligosaccharides to carbohydrases such as a-amylase, aglucosidase, glucoamylase, dextranase, and ~-fructofuranosidase to find special substrates for discriminative enzyme assays. In this paper, we deal with p r e p a r a t i o n o f leucrose from sucrose with or without fructose using dextransucrase and enzymatic hydrolysis o f leucrose. Enzyme (1.5 units per ml; specific activity, 5.8 u / r a g protein) was prepared from a culture filtrate o f Leuconostoc mesenteroides by the method reported previously (11). Dextransucrase activity was assayed as follows. One-half ml o f a solution o f enzyme was a d d e d to 0.5 ml of 200/00 sucrose solution containing 0.05 M acetate buffer, p H 5.2, at 30°C. A f t e r a one-hour incubation, the reducing sugar f o r m e d was measured as fructose by the method of Somogyi and Nelson (12). One unit of the enzyme activity was defined as the a m o u n t o f enzyme that converts 1 ~tmol o f sucrose into fructose and dextran per minute under the above conditions. Oligosaccharides were synthesized from sucrose alone (400 mg), or sucrose (200 mg) and fructose (200 mg), by incubating with 15 units o f dextransucrase in a solution of 0.05 M acetate buffer, p H 5.2, at 30°C for 40 h and were isolated after column c h r o m a t o g r a p h y on Bio Gel P-2 (3 × 120 cm). The p r o d u c t i o n o f (V) by dextransucrase was greatly increased with fructose as the acceptor o f the glucosyl residue. The yield o f (V) was 38.80//00, based on sucrose used as the substrate in the case o f the reaction done with fructose, while the yield was only 11.3°//00 t o w a r d sucrose substrate alone. Analyses o f p r o d u c t (V) were done by Thin Layer Chrom a t o g r a p h y (TLC) using a silica gel 60 (Merck) plate (20 × 20 cm) as the adsorbent with a solvent system o f n* Corresponding author. 277

278

IIZUKA ET AL.

J. FERMrNT. BIOENG.,

TABLE 1. Oligosaccharides and dextran synthesized from sucrose with or without fructose by dextransucrase of L. mesenteroides Substrate Sucrose (400 rag) Sucrose-- (200 mg) "~ Fructose (200 rag);

Saccharide Tetra (III)Tri (IV)-

Dextran (I)

Penta (II)-

183.0

8.3

9.4

31.8

12.7

19.4

Di (V)-

Reducing sugar (VI) (Fru +Glu)

8.3

44.6

135.3

24.1

77.4

196.6

The yield was expressed in terms of weight (mg) of product.

i00

~e

e

e,

"-

11-4' d

a

50

J; i00

w

l -o'

v

;

I/--o

b

50

i00

.a.A

A

f

C

/

~0

?

50

F

0

0.5

1

2

~

4

24

0

0.5

0

®

®

1

2

4

f 24

Reaction time (h) FIG. 1. Hydrolysis of maltose, sucrose, and leucrose with carbohydrases. The reaction mixture contained 0.5 mg of substrate and I unit of a-glucosidase. The reaction was done at 37°C except the reaction with yeast a-glucosidase which was done at 30°C. Symbols: o , maltose; ±, sucrose; ©, leucrose, a, Porcine serum a-glucosidase; b, bovine serum a-glucosidase; c, human urinary a-glucosidase; d, yeast ,-glucosidase; e, Rhizopus glucoamylase; f, Candida fl-fructofuranosidase.

bacterial a-amylase, Rhizopus glucoamylase, Chaetom i u m d e x t r a n a s e , and C a n d i d a f l - f r u c t o f u r a n o s i d a s e did n o t p r o d u c e a d e t e c t a b l e a m o u n t o f r e d u c i n g sugar d u r i n g i n c u b a t i o n for 24 h, as s h o w n in Fig. 1. F u r t h e r m o r e , the a m o u n t o f the r e a c t i o n p r o d u c t s f r o m leucrose w i t h an e n z y m e m i x t u r e o f g l u c o a m y l a s e a n d b o v i n e s e r u m c~-glucosidase was a l m o s t the s a m e as with b o v i n e s e r u m a - g l u c o s i d a s e a l o n e (Fig. 2). This indicates that leucrose can be used as a substrate for detection or measurem e n t o f a - g l u c o s i d a s e in e n z y m e m i x t u r e s such as culture filtrates o f m i c r o o r g a n i s m s . T h e difference in activity t o w a r d leucrose with a - g l u c o s i d a s e r e m a i n e d in future study.

REFERENCES 1. Jeanes, A. (R)., Haynes, W. C., Wiiham, C. A., Rankin, J. C., Melvin, E.H., Austin, M.J., Cluskey, J.E., Fisher, B.E., Tsuehiya, H. M., and Rist, C. E.:Characterization and classification of dextrans from ninety-six strains of bacteria. J. Amer. Chem. Soc., 76, 5041-5052 (1954). 2. Jeanes, A. (R)., Wilham, C. A., and Miers, J. C.: Preparation and characterization of dextran from Leuconostoc mesenteroides. J. Biol. Chem., 176,603-615 (1948). 3. Wilham, C. A., Alexander, B.H., and Jeanes, A. (R).: Heterogeneity in dextran preparations. Arch. Biochem. Biophy., 59, 61-75 (1955). 4. Koepseil, H. J. and Tsuehiya, H. M.: Enzymatic synthesis of dextran. J. Bacteriol., 63, 293-295 (1952). 5. Toroi, R. Hayashi, J., Ohara, N., and Kozaki, M.: Oligosaccharide production by dextransucrase of Streptococcus boris

VOL 70, 1990

•~

NOTES

If~

I00

9.

r. 0

10. SO g4 0 ID ID

11.

'o'.s

Reaction time (h)

"4

2'4

FIG. 2. Hydrolysis of maltose and leucrose with the enzyme mixture of bovine a-glucosidase and Rhizopus glucoamylase. The reaction was carried out at 37°C. Enzyme and substrate used, glucoamylase (1 unit) and a-glucosidase (1 unit); 0.5 mg substrate. Symbols: o , maltose; ©, leucrose. no. 148 isolated from bovine rumen. Nippon Shokuhin Kogyo Gakkaishi, 37, 355-362 (1990). 6. Bourne, E. J., Hutson, D. H., and Weigel, H.: Oligosaccharides in dextran-producing cultures of Streptococcus boyle. Biochem. J., 79, 549-553 (1961). 7. Bailey, R.W., Baker, S.A., Bourne, E.J., Grant, P. M., and Stacey, M.: Synthesis of oligosaccharides by growing cultures of Betacoccus arabinosaceous. Nature, 175, 635 (1955). 8. Barker, R. W., Bourne, E. J., Grant, P. M., and Stacey, M.: Immunopolysaccharides. IX. The enzymic synthesis of trisaccha-

12. 13.

14.

15. 16. 17.

279

rides containing the a-l,2-glucosidic linkage. J. Chem. Soc., 1895-1902 (1958). Irlki, Y. and Hehre, E . J . : The scope of interanomeric glycosyl transfer reactions: hetero-dialdoside synthesis by enzymic glucosylation of D-galactose and o-mannose. Arch. Biochem. Biophys., 134, 130-138 (1969). Robyt, J. F. and Eklund, S. H.:Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydr. Res., 121, 279-286 (1983). Itaya, K. and Yamamoto, T.: Dextransucrase as an enzyme associating with alkaline earth metal ions. Agric. Biol. Chem., 39, 1187-1192 (1975). Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). Jarrell, H. C., Conway, T. F., Moyana, P., and Smith, 1. C. P.: Manifestation of anomeric form ring structure, and linkage in the ~3C-NMR spectra of oligomers and polymers containing Dfructose: maltulose, isomaltulose, sucrose, leucrose, 1-kestose, nystose, inulin and grass levan. Carbohydr. Res., 76, 45-57 (1979). Hibi, H., Chiba, S., and Shimomura, T.: Purification and some properties of a neutral a-glucosidase from pig serum. Agric. Biol. Chem., 40, 1805-1812 (1976). Minamiura, N., Chiura, H., Tsujino, K., and Yamamoto, T.: Glucose-forming amylase in human urine. J. Biochem., 77, 1015-1022 (1975). lizuka, M., Tsuji, Y., and Yamamoto, T.: Some physicochemical properties of Candida utilis. Agric. Biol. Chem., 38, 213-215 (1974). Hattori, A., Ishibashi, K., and Minato, S.: The purification and characterization of the dextranase of Chaetomium gracile. Agric. Biol. Chem., 45, 2409-2416 (1981).