Mikihiko Kobayashi

Purification and properties of a novel enzyme from Bacillus spp. T-3040, which catalyses the conversion of dextran to cyclic isomaltooligosaccharides

A novel enzyme, cycloisomaltooligosaccharide glucanotransferase (CITase), catalyzes the conversion of dextran to cyclic isomaltooligosaccharides by intramolecular transglucosylation (cyclization reaction). CITase was purified to homogeneity from the culture filtrate of Bacillus sp. T‐3040 isolated from soil. The M r of the enzyme was estimated to be 98,000 by SDS‐PAGE. The enzyme catalyzed the cyclization reaction and gave three cyclic isomaltooligosaccharides (cycloisomalto‐heptaose, ‐octaose, and ‐nonaose) at a total yield of about 20%. Coupling and disproportionation reactions were also observed. These results showed that this enzyme is a multi‐functional enzyme which catalyzes intramolecular and intermolecular transglucosylation.

Novel Cyclic Dextrins, Cycloisomaltooligosaccharides, from Bacillus sp. T-3040 Culture.

Three kinds of novel cyclic isomaltooligosaccharides were isolated from the culture broth of a strain of Bacillus sp. T-3040, which had been isolated from soil. They were identified as cycloisomalto-heptaose, -octaose, and -nonaose, respectively, on the basis of their mass spectra, proton nuclear magnetic resonance spectra, carbon nuclear magnetic resonance spectra, infrared spectra, reducing power, and results of enzymatic analysis using endo-dextranase and exo-dextranase. They also showed features similar to cyclodextrins upon high-pressure liquid chromatography analysis using an ODS column.

Water-soluble and water-insoluble glucans produced by Escherichia coli recombinant dextransucrases from Leuconostoc mesenteroides NRRL B-512F

Two dextransucrase genes, dsrS and dsrT5, from Leuconostoc mesenteroides NRRL B-512F were expressed in Escherichia coli, and recombinant dsrT5 dextransucrase was shown to produce a water-insoluble glucan. In contrast, native dextran from L. mesenteroides B-512F is water-soluble. The water-insoluble glucan was shown by 13C NMR and glycosyl-linkage composition analysis to contain about 50% 6-linked Glcp and 40% 3-linked Glcp. The primitive B-512F strain is suggested to have produced water-insoluble glucan containing 3-linked Glcp. The glucans produced by dextransucrases expressed in E. coli contained 4-linked Glcp, as shown by glycosyl-linkage composition analysis. The amount of 4-linked Glcp was increased when the truncated, water-insoluble, glucan-producing dextransucrase, which does not have C-terminal repeating units, was added to the water-soluble, glucan-producing dextransucrase. Trace amounts of 4-linked Glcp were also detected in the dextran obtained from the B-512F culture supernatant, in dextran produced by dextransucrase purified from the B-512F strain culture supernatant, and in clinical dextran. The results of glycosyl-linkage composition analysis suggest that dextransucrases produce 4-linked Glcp as well as 6- and 3-linked Glcp.

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene, and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H.K. Kuramitsu, Plasmid 34 (1995) 85-95. This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb Bg/II fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.

Utilization of NaCl to suppress the growth of contaminating microbes during the continuous production of cycloisomaltooligosaccharides by immobilized enzyme

Abstract Cycloisomaltooligosaccharide glucanotransferase (CITase) catalyzes the synthesis of cycloisomaltooligosaccharides (CIs) from dextran. As the immobilized CITase was unstable at high temperature, microbial contamination during continuous CI production is a serious problem. In this study, the utilization of NaCl to suppress the growth of contaminating microbes was examined. Surprisingly, free CITase was very tolerant against NaCl and the enzyme activity was not influenced by up to 5xa0mol/l (29.2%) NaCl. The activity of the enzyme immobilized on Chitopearl BCW-3505 was not influenced by up to 2xa0mol/l (11.7%) NaCl. When 2xa0M NaCl was included in the substrate solution during continuous production of CIs by a column system packed with the immobilized enzyme, no microbial contamination appeared and CIs were produced for 40 days. The added NaCl had no influence on the life time of the system and was effective in suppressing the growth of contaminating microbes. The added NaCl could be separated easily from CIs by an open column system packed with activated carbon. Although it is well known that NaCl has a suppressive effect on the growth of microbes, to our knowledge, this is the first report on utilization of NaCl for the production of saccharides.