Isolation and purification of Flavobacterium α 1,3 glucanase hydrolyzing, insoluble, sticky glucan of Streptococcus mutans

Abstract

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell free extracts of S. mutans, S. sanguis, and S. sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek 14 bacterium. EK 14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl cellulose (DE 32) column and gradient elution chromatography with a carboxymethyl cellulose (CM 32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the α 1,3 glucan endolytically and was inactive toward glucans containing α 1,6, α 1,4, β 1,3, β 1,4, and/or β 1,6 bonds as the main linkages.