Hidehiko Mukasa

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g)

In addition to the 1,3‐α‐D‐glucan synthetase (pI 4.9) and the highly‐branched 1,6‐α‐D‐glucan synthetase (pI 3.9–4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (pI 5.5–7.0), which exhibited 87% 1,6‐α‐bond‐, 6% 1,3‐α‐bond‐ and 7% 1,3,6‐branch‐forming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween 80‐supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.

Purification and properties of Streptococcus mutans extracellular glucosyltransferase.

Extracellular glucosyltransferase (sucrose:1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) was purified about 10 000-fold from the culture supernatant of Streptococcus mutans 6715. The enzyme preparation was homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing and ultracentrifugation analyses. The specific activity of the enzyme was 34.9 I.U. per mg of protein and the carbohydrate content was less than 1% (w/w). The molecular weight was determined to be 149 000 +/- 5000 by sedimentation equilibrium experiment. The acidic and basic amino acids of the enzyme comprised 29 and 8.4% of total amino acid, respectively, and the isoelectric point was pH 4.1. The enzyme had the optimum pH of 5.5 and the Km value of 2.4 mM for sucrose. The water-soluble glucan, which was de novo-synthesized from sucrose by the purified enzyme, was analyzed by a gas-liquid chromatography-mass spectroscopy and was found to be 1,6-alpha-D-glucan with highly (35%) branched structure of 1,3,6-linked glucose residue.

Direct activity stains for glycosidase and glucosyltransferase after isoelectric focusing in horizontal polyacrylamide gel layers

Abstract Ampholytes up to 2% (w/v) did not significantly inhibit enzymatic activities of β-fructofuranosidase, α-amylase, dextranase, and Streptococcus mutans glucosyltransferase. Based on this finding, rapid activity stain methods were developed to locate these enzymes in polyacrylamide gel layer without any efforts to remove carrier ampholytes after isoelectric focusing. The reducing sugars and glucan accumulated in gel during direct incubation after focusing were stained with triphenyltetrazolium reagent and with periodic acid-Schiffs reagent, respectively. The activity stains for β-fructofuranosidase and glucosyltransferase were highly sensitive and specific, compared with the corresponding protein stain. In the cases of α-amylase and dextranase for which substrates were starch and dextran polymers, a thinner gel layer was especially required. Additionally, several technical modifications are described in order to operate these focusing and staining procedures rapidly and successively.

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.

Comparative Study of Streptococcus mutans Extracellular Glycosyltransferases by Isoelectric Focusing

Extracellular glycosyltransferases from 17 strains of Streptococcus mutans were examined by analytical isoelectric focusing. Three kinds of glucosyltransferase: highly-branched-1,6-alpha-D-glucan synthetase, 1,3-alpha-D-glucan synthetase and 1,6-alpha-D-glucan synthetase, were excreted from serotype a, d and g strains. The enzymes of serotype a strains were distinguishable from those of serotypes d and g by differences in their pI values. Serotype c, e and f strains excreted basic glucosyltransferase and acidic fructosyltransferase. Serotype b strains also excreted the glucosyl- and fructosyltransferases, but the pI values were different from those of the enzymes from the other serotypes. Thus, S. mutans strains could be divided into four groups by analytical isoelectric focusing of glycosyltransferases which corresponded well to the four genetic groups.

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan.

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.

Purification and Properties of Extracellular Glucosyltransferase Synthesizing 1,6-, 1,3-α-D-Glucan from Streptococcus mutans Serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.

Purification and Characterization of Cell-associated Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans Serotype c

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.

Purification and properties of extracellular glucosyltransferases from Streptococcus mutans serotype a

Extracellular glucosyltransferases (sucrose: 1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6.20 and 5.86 i.u. mg-1, respectively, exhibited slightly different isoelectric points (pI 4.5 and 4.2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5.5 and the same Km values of 1.3 mM for sucrose and of 83 microM-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-alpha-D-glucans with 20 and 24.5 mol% 1,3,6-branch points, respectively. Both are therefore bifunctional enzymes.

Nigerooligosaccharide acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I.

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.