Purification of glucosyltransferases (GtfB/C and GtfD) from mutant strains of Streptococcus mutans.

Abstract

Streptococcus mutants constitutively expresses three glucosyltransferases (GTFs), i.e., GtfB, GtfC, and GtfD, which synthesize glucan polymers from sucrose. Two genetically constructed mutants of S. mutans which stably expressed either the cell-associated or the extracellular GTFs were selected for purification and characterization of these enzymes. The cell-associated GtfB and GtfC from strain GS-5DD lacking the gtfD gene expression were extracted by urea, renatured by dialysis in sodium phosphate buffer and then separated from the other wall-associated components by column chromatography. The extracellular GtfD was purified from the culture supernatant of strain NHS1 lacking gtfB and gtfC gene expression. The molecular weights of the purified GTFs was similar (150-160 kDa), as determined by SDS-polyacrylamide gel electrophoresis. The GtfB/C preparation synthesized primarily water-insoluble glucan in a primer independent manner. However, the presence of the dextran enhanced the enzymatic activities of the GtfB/C. GtfD synthesized water-soluble glucan exclusively in a primer dependent manner. Purified GtfD had a pH optimum of 5.5, and a K(m) value of 4.35 mM for sucrose. These results indicated that the mutated strains served as an efficient and specific host to obtain native GTFs.