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Preparation of glucosyl transferase ec from streptococcus mutans by evolution from water insoluble poly saccharide with a dissociating solvent

, : Preparation of glucosyl transferase ec from streptococcus mutans by evolution from water insoluble poly saccharide with a dissociating solvent. Infection and Immunity 23(2): 446-452

Glucosyltransferase [GTF] (EC 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 S. mutans strain 6715. Gel filtration of the GTF 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 GTF preparation contained 22% carbohydrate and eluted at a position corresponding to a MW of 422,000. Polyacrylamide gel (5%) electrophoresis of this preparation revealed 2 regions which stained for protein, formed water-insoluble polysaccharide in the presence of sucrose, and precipitated with [hamster] antisera directed to crude GTF preparations. The guanidine-eluted enzyme could be primed by 5 .times. 10-5 M dextran T10 (MW 10,000). High-MW glucan, a possible glucan-binding protein and GTF were obtained after the final gel filtration step (4% cross-linked agarose). [The ability to obtain GTF free of other antigens (Ag) in a yield sufficient for immunization experiments may help resolve the role of GTF as a protective Ag in vaccines.].

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