Preparation and Properties of a β-d-Glucanase for the Specific Hydrolysis of β-d-Glucans

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Abstract
A .beta.-D-glucanase [EC 3.2.1.73] highly specific for glucans containing a linkage sequence.cntdot..cntdot..cntdot.Glc 1 .fwdarw. 4 Glc 1 .fwdarw. 3 Glc 1 .fwdarw. 4 Glc.cntdot..cntdot..cntdot.was isolated from several commercial preparations of Bacillus subtilis .alpha.-amylase including one purified by repeated crystallization. The .beta.-D-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the .alpha.-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing suqars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity of pH 6.5, a Vmax of 13.9 .mu.g glucose equivalent released/.mu.g protein .cntdot. h and an apparent Km of 3.4 mg of lichenan per ml. Potential applications of this enzyme for the structural characterization of plant cell wall glucans is discussed.