Lysis of yeast cell walls: glucanases from Bacillus circulans WL-12.

Endo-beta-(1 --> 3)- and endo-beta-(1 --> 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-beta-(1 --> 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-beta-(1 --> 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-beta-(1 --> 3)-glucanase was specific for the beta-(1 --> 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. beta-(1 --> 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-delta-lactone was a noncompetitive inhibitor and Hg(2+) inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified beta-(1 --> 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified beta-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.