Isolation and characterization of an extracellular glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c

Abstract

A glucosyltransferase which synthesized insoluble glucan in polyacrylamide gel was isolated from the culture supernatant of Streptococcus mutans Ingbritt (serotype c) by ultrafiltration, ethanol fractionation, isoelectric focusing, and preparative gel electrophoresis. The enzyme preparation was electrophoretically homogeneous and immunologically distinct from the highly branched 1,6-alpha-D-glucan synthase and fructosyltransferase from the same strain and glucosyltransferases from serotypes a and g. The molecular weight was 99,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was 8.5. The enzyme had the optimum pH of 6.0 and Km value for sucrose of 9.4 mM. Besides the insoluble glucan with 96% 1,3-alpha linkage, this enzyme synthesized a considerable amount of diffusible glucan with 84% 1,6-alpha linkage, separately. This enzyme may be the one released from the enzyme aggregates by extracellular proteases, because the addition of extraneous trypsin to the crude enzyme preparation increased the amount of the enzyme species.

Transcriptional repressor Rex is involved in regulation of oxidative stress response and biofilm formation by Streptococcus mutans

“…To measure the extracellular glucose polymers in biofilms, a phenol-sulfuric acid assay was used with known concentrations of glucose as the standards (Mukasa et al, 1985;Kumada et al, 1987;Ausubel et al, 1992;Werning et al, 2008). Briefly, 3-day biofilms were grown in BMGS on glass slides in 50-mL tubes as described elsewhere (Phan et al, 2000;Wen et al, 2010a, b).…”

Nasal immunization of humans with dehydrated liposomes containing Streptococcus mutans antigen

“…Protein concentration was determined with a Coomassie protein reagent using bovine serum albumin as standard (Pierce Chemical Co., Rockford, IL). The protein fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% acrylamide gels) (31) analyses, and the relative purity and enzymatic activity of the GTF was determined by Coomassie stain and periodic acid Schiff (PAS) stain following incubation of the gel with sucrose, as described by Mukasa et al (37).…”

Properties of practical oral liposome‐Streptococcus mutans glucosyltransferase vaccines for effective induction of caries protection

“…Protein concentration was determined using the Pierce Coomassie Protein reagent and a bovine serum albumin standard (Pierce Chemical Co., Rockford, IL). The protein fraction was subjected to SDS-PAGE (7.5% acrylamide gel) analysis (17), and the purity and enzymatic activity of the GTF was determined by Coomassie stain and periodic acid Schiff (PAS) stain following incubation of the gel with sucrose, as described by Mukasa et al (30).…”