Lysis of Yeast Cell Walls Lytic β‐(1→6)‐Glucanase from Bacillus circulans WL‐12

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Abstract
When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.