Purification and Some Properties ofp-Nitrophenyl-β-D-glucoside-hydrolyzing Enzymes in Culture Filtrate ofBacillus circulansKA-304 Grown on Cell-wall Preparation ofSchizophyllum commune

Hydrolyzing activities toward p-nitrophenyl (p-NP)-β-D-glucoside and laminarin in a culture filtrate of Bacillus circulans KA-304, which has been observed to form protoplasts from Schizophyllum commune mycelia, increased when the bacterium was grown on a cell-wall preparation (CWP) of S. commune or laminarin as a carbon source. An analysis of the filtrate with the CWP suggested occurrence of two major p-NP-β-D-glucoside-hydrolyzing enzymes (β-D-glucosidases I and II) and a laminarin-hydrolyzing enzyme. After separation by DEAE-cellulose column chromatography, β-D-glucosidases I and II were isolated (β-D-glucosidase I: 13-fold purification with 34% yield; β-D-glucosidase II: 26-fold with 8%). The enzymes resembled each other in their properties except for their molecular weight, subunit structure (β-D-glucosidase I: 200,000, tetramer; II: 100,000, dimer), and susceptibility to such substances as p-chloromercuribenzoic acid and Ag(+) ion. β-D-Glucosidases I and II hydrolyzed gentiobiose (β-1,6 glucosidic linkage; Km=3.6 mM, β-D-glucosidase I; 4.6 mM, β-D-glucosidase II) and laminaribiose (β-1,3 glucosidic linkage; Km=6.1 mM, β-D-glucosidase I; 6.7 mD β-D-glucosidase II), and showed a certain reactivity toward laminarin as well.