Preparation of glucosyltransferase from Streptococcus mutans by elution from water-insoluble polysaccharide with a dissociating solvent.
Glucosyltransferase (EC 2.4.1.5) was obtained by dissociation from water-insoluble polysaccharide in the presence of 6 M guanidine-hydrochloride. Water-insoluble polysaccharide was synthesized by cell-free culture supernatants from Streptococcus mutans strain 6715. Gel filtration of the glucosyltransferase on a column of 8% agarose in phosphate buffer, followed by filtration on a column of 4% cross-linked agarose in 6 M guanidine-hydrochloride, gave a 23-fold enrichment of the enzyme. The enriched glucosyltransferase preparation contained 22% carbohydrate and eluted at a position corresponding to a molecular weight of 422,000. Polyacrylamide gel (5%) electrophoresis of this preparation revealed two regions which stained for protein, formed water-insoluble polysaccharide in the presence of sucrose, and precipitated with antisera directed to crude glucosyltransferase preparations. The guanidine-eluted enzyme could be primed by 5 X 10(-5) M dextran T10 (molecular weight, 10,000). High-molecular-weight glucan and a possible glucan-binding protein were also obtained after the final gel filtration step (4% cross-linked agarose) in addition to glucosyltransferase.
D002850:Chromatography, Gel; D003911:Dextrans / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006146:Guanidines; D008722:Methods; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis; D013295:Streptococcus mutans / Q000201:enzymology*
D003911:Dextrans; D006146:Guanidines; D011135:Polysaccharides, Bacterial; D005964:Glucosyltransferases