J. Gen. App!. Microbiol., 34, 213-219 (1988) ON THE DIFFERENCE IN ADSORPTION ON SEPHADEX GEL OF THE DEXTRANSUCRASE OF STREPTOCOCCUS BOVIS GROWN ON SUCROSE AND GLUCOSE MEDIA TOSHIRO HAYASHI, RYO IOROI,* NAOHIRO OHARA,* AND MICHIO KOZAKI* Mejiro-gakuen Women's College, Shinjuku-ku, Tokyo 160, Japan * Tokyo University of Agriculture, Setagaya-ku, Tokyo 156, Japan (Received September 16, 1987) The dextransucrase produced by orange-colored Streptococcus bovis No. 148 isolated from bovine rumen was investigated. The enzyme was produced in both sucrose and glucose media. The enzyme prepared from the glucose medium was specifically adsorbed on various Sephadex gels. But that from the sucrose medium was not adsorbed. When the enzyme from the glucose medium was incubated with a certain dextran, it was no longer adsorbed on Sephadex. There was no saccharide in the enzyme produced in glucose medium. Dextransucrase is produced in sucrose medium by Leuconostoc mesenteroides. It converts sucrose into dextran and fructose (1). Dextran is a glucose polymer composed mostly of a-1,6-glucosidic linkages, and its molecule is an ordinary highly branched structure with a-1,3, a-1,2-glucosidic linkages. The degree of branching varies with the strain of organism (2). The reason for this is not clear. Theories about the formation of the branching linkage of dextran have been proposed as the branching enzyme theory (3, 4), the acceptor dependence theory (5), and others (6, 7). To determine the reaction mechanism of the dextransucrase, the pure enzyme must be prepared free from preformed dextran. Recently, TsUMURAYA et al. (8) indicated that highly purified enzyme still contained small amounts of dextran, and that dextran is not simply a contaminant, but forms a complex with the enzyme. BAILEY (9) found that Streptococcus bovis isolated from bovine rumen produced dextransucrase in both sucrose and glucose media. He also found that the saccharide content in the enzyme produced in the glucose medium is low compared to that produced in the sucrose medium. Address reprint requests to: Dr. T. Hayashi, Mejiro-gakuen Women's College, 4-chome, Nakaochiai, Shinjuku-ku, Tokyo 160, Japan. 213
214 HAYASHI, IOROI, OHARA, and KOzAKI VOL. 34 In the course of studies on dextransucrase we have found that the enzyme produced by the orange-colored S. bovis in glucose medium does not contain any saccharide, and is specifically adsorbed on Sephadex gel. This paper describes the enzyme production of orange-colored S. bovis and the adsorption of the enzyme on Sephadex gel. MATERIALS AND METHODS Organism. Orange-colored Streptococcus bovis No. 148 was used as the test strain. Leuconostoc mesenteroides NRRL B-512F was used as the reference strain. Medium. The basal medium used in the present study is shown in Table 1. Culture. The culture of S. bovis was maintained by the anaerobic culture method previously reported (10). L, mesenteroides was grown using the method of TSUCHIYA et al. (1). Cell growth was monitored by absorbance at 660 nm. Assay of dextransucrase. Dextransucrase activity was measured by monitoring the release of reducing sugar during incubation of the enzyme with sucrose as substrate, since has been found no enzyme activity that releases reducing sugar from sucrose except the dextransucrase from the culture fluid of S. bovis and L. mesenteroides. Cultures were centrifuged at 25,000 x g for 20 min and the supernatant was dialyzed against DW. The dialyzates were used as an enzyme preparation. The reaction mixture contained 1 ml of enzyme preparation, 4 ml of 0.05 M acetate buffer (ph 5.5), and 300 mg of sucrose. Reducing sugar (fructose) was measured by the method of SHAFFER and SOMOGYI (I1). One unit of dextransucrase activity was defined as the level of enzyme activity which produced 0.52 mg of fructose per ml of reaction mixture in 1 hr at 40 C (S. bovis) or 30 C (L. mesenteroides). Adsorption of enzyme on Sephadex gel. Organisms were grown in 2 % sucrose or 4% glucose PY media. The cell-free culture fluid was evaporated 8-fold at ph 5.5 under vacuum (below 37 C) and dialyzed against running water for 12 hr. Table 1. Composition of culture medium.
1988 S. bovis Dextransucrase 215 Ammonium sulfate was added to the dialyzate until it was 80 c saturated. The precipitate formed was collected by centrifugation (15,000 x g, 15 min) and dissolved in a small quantity of distilled water. Insoluble materials were removed by centrifugation (25,000 x g, 15 min), and stored at 4 C. Two ml (about 2,000 units as dextransucrase) of the enzyme preparation was placed on a Sephadex gel column (25 x 200 mm) equilibrated with 0.05 M phosphate buffer (ph 5.5), then eluted with 500 ml of the same buffer at a velocity of 40 ml/hr. The adsorbed enzyme on Sephadex gel was determined by subtracting the amount of enzyme eluted (nonadsorbed enzyme) from the enzyme placed on the column. The Sephadex columns (Pharmacia Fine Chemicals, Uppsala, Sweden) used were G-25, G-50, G-75, G-100, and G-200. Recovery of adsorbed enzyme from Sephadex gel. The Sephadex G-200 with its adsorbed enzyme (about 1,900 units) was poured into a 300-ml beaker from a column with 50 ml of 0.05 M phosphate buffer (ph 5.5) added, then left to stand for 3 hr at various temperatures. The mixture was then filtered through glass filters 3G3. Protein assay. Protein was determined by reading the absorbance at 280 nm, using bovine serum albumin as the reference standard. Carbohydrate assay. Carbohydrate was determined by the phenol-sulphuric acid method (12), using anhydrous D-glucose as the reference standard. RESULTS AND DISCUSSION Time course of enzyme production The growth and enzyme production curves of orange-colored Streptococcus bovis No. 148 in sucrose PY medium are shown in Fig. 1. Dextransucrase was produced while the bacteria were growing and the production was reduced after the growth stopped. When the culture medium was not neutralyzed with NaHC03, the enzyme was rapidly inactivated (data not shown). Influence of sugars on enzyme production Table 2 shows the effect of sugars on enzyme production of S. bovis and Leuconostoc mesenteroides NRRL B-512F in PY medium. Sucrose was the most effective carbon source for enzyme production. In S. bovis, glucose was also effective in the production. BAILEY (9) reported that dextransucrase in S. bovis may be a constitutive enzyme rather than an adaptive one, since the bacterium could produce the enzyme in the absence of sucrose. However, the dextransucrase was produced only when sucrose or glucose was present in the medium, as shown in Table 2. Therefore, the enzyme of S. bovis may be an adaptive one. Adsorption of the enzyme on Sephadex gel When the enzyme produced in glucose medium was applied on a column the recovery of the enzyme (G-DS) was extremely low compared with that produced in
216 HAYASHI, IORoI, OHARA, and Koz uu VOL. 34 Fig. 1. Time courses of the dextransucrase production and the growth of S. bovis. o, absorbance (OD66onm); o, dextransucrase activity. S. bovis was incubated in the PY medium supplemented with 2 % sucrose, and neutralyzed by NaHCO3. Table 2. Influence of various carbohydrates on the dextransucrase production by S. bovis and L. mesenteroides. sucrose medium (S-DS) (Table 3). More than 80% of the G-DS was bound to the gel except in the Sephadex G-25 column (Table 4). It was calculated that about 180,000 units of enzyme was bound to 1 g of Sephadex G-200. Sephadex is produced by the reaction between dextran and epichlorohydrin, and the molecules are heavily entangled with glyceryl bridges. Sephadex G-25 is the most heavily cross-linked of the Sephadex types tested (13). The low enzyme adsorption of Sephadex G-25 may be caused by heavy cross-linkages. The batchwise adsorption of the G-DS was also tested. However, the enzyme adsorbed on Sephadex G-200 was less than 60 c in all tests. In the early stages of enzyme adsorption, the Sephadex and enzyme may be in equilibrium. Table 5 shows the effect of various saccharides on the adsorption of the G-DS in Sephadex gel. Mixtures of the enzyme and various saccharide solutions were
1988 S. bovis Dextransucrase 217 Table 3. Adsorptions of dextransucrase produced by S. b ovis L, mesenteroides on Sephadex gel. and Table 4. Adsorption on Sephadex gels of S. bovis produced in glucose PY medium. dextransucrase Table 5. Influence of various saccharides on the adsorption of S. b ovis dextransucrase on Sephadex gel G-200 column.
218 HAYASHI, IOROI, OHARA, and KozAKI VOL. 34 Table 6. Effect of various eluents for the recovery of adsorbed dextransucrase on the Sephadex eel G-200 column. Fig. 2. Effect of temperature for the release of adsorbed dextransucrase on Sephadex gel G-200. Enzyme-Sephadex gel complex was left to stand with 0.05 M phosphate buffer (ph 5.5) for 3 hr at various filters. 41 V I V ~ ~, temperatures, then filtered through glass applied on a Sephadex gel column at 4 C, then eluted with 500 ml of 0.05 M phosphate buffer (ph 5.5) at a velocity of 40 ml/hr. The adsorption was greatly decreased by dextran, lactose, and glucose. These results suggest that, unlike S-DS, G-DS contains little dextran or other saccharides. Substantial quantity of the enzyme adsorbed on Sephadex gel column was eluted with a dextran solution at a velocity of 60 ml/hr, 4 C (Table 6), and the eluted enzyme combined with the dextran to form an enzyme-dextran complex. About 2l of 0.5 0 dextran T-2000 solution was needed to elute 80 c of enzyme adsorbed in the Sephadex gel. Adsorption of the enzyme in Sephadex gel was inhibited by glucose or sucrose, but these sugars had no effect on the elution of the Sephadex-bound enzyme. The enzyme adsorbed on Sephadex was also released by incubating the
1988 S. bovis Dextransucrase 219 enzyme-sephadex complex. As shown in Fig. 2, about 60 c of the adsorbed enzyme was released when the gel was incubated with 0.05 M phosphate buffer (ph 5.5) at 40 C for 3 hr. The enzyme, released by the incubation, was concentrated about 10- fold by evaporation under vacuum, and the carbohydrate was determined by the phenol-sulphuric acid method. Carbohydrate could not be detected in the enzyme preparation. The result shows that G-DS recovered from Sephadex gel may not contain any carbohydrates, the enzyme may therefore be saccharide-free protein. The authors thank Dr. Tai Utimura of Tokyo University of Agriculture for many useful suggestion. REFERENCES 1) H. M. TSUCHIYA, H. J. KOEPSELL, J. CORMAN, G. BRYANT, M. O. BOGARD, V. H. FEGER, and R. W. JACKSON, J. Bacteriol., 64, 521 (1952). 2) A. JEANES, W. C. HAYNES, C. A. WILHAM, J. C. RANKIN, E. H. MELVIN, M. J. AUSTIN, J. E. CLUSKEY, B. E. FISHER, H. M. TSUCHIYA, and C. E. RIST, J. Am. Chem. Soc., 76, 5041 (1954). 3) R. W. BAILEY, S. A. BARKER, E. J. BOURNE, and M. STACEY, J. Chem. Soc., p. 3530 (1957). 4) F. A. BoVEY, J. Polym. Sci., 35, 169 (1959). 5) K. H. EBERT and F. PATAZ, Z. Naturforsch., 17b, 738 (1962). 6) D. SUZUKI and T. KOBAYASHI, Agric. Biol. Chem., 39, 557 (1975). 7) M. KoBAYASHI and K. MATSUDA, Agric. Biol. Chem., 41, 1931 (1977). 8) Y. TSUMURAYA, N. NAKAMURA, and T. KoBAYASHI, Agric. Biol. Chem., 40, 1471 (1976). 9) R. W. BAILEY, Biochem. J., 72, 42 (1959). 10) T. HAYASHI and K. KITAHARA, J. Gen. Appl. Microbiol., 22, 301 (1976). 11) P. A. SHAFFER and M. S0M0GYI, J. Biol. Chem., 100, 695 (1933). 12) M. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, and F. SMITH, Anal. Chem., 28, 350 (1956). 13) P. FLORIN, Dextran Gels and Their Application in Gel Filtration, AB Pharmacia, UPPSALA, Sweden (1962), 85 pp.