Purification and characterization of Streptococcus mutans glucosyltransferase (GtfC) expressed in Escherichia coli

Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.