Purification and some properties of a beta-glucosidase from Flavobacteriumjohnsonae

Flavobacterium johnsonae was isolated as microorganism that produced a beta -glucosidase with hydrolytic activity of beta-glucosyl ester linkages in st eviol glycosides. The enzyme was purified to homogeneity from a cell-free e xtract by streptomycin treatment, ammonium sulfate fractionation, and colum n chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point o f pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature w as 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme h ydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at s ite 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta- glucosides such as p-nitrophenyl beta-glucoside, phenyl beta-glucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme a ctivity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2 +, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual a ctivity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost th e same degrees. Kinetic behaviors in the mixed substrate reactions of rebau dioside A and steviol monoside, and of steviol monoglucosyl ester and pheny l beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.