PURIFICATION AND SOME PROPERTIES OF P-NITROPHENYL-BETA-D-GLUCOSIDE-HYDROLYZING ENZYMES IN CULTURE FILTRATE OF BACILLUS-CIRCULANS KA-304 GROWN ON CELL-WALL PREPARATION OF SCHIZOPHYLLUM-COMMUNE

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