Purification and Some Properties of a β-Glucosidase from Flavobacterium johnsonae

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Flavobacterium johnsonae was isolated as a microorganism that produced a β-glucosidase with hydrolytic activity of β-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72kDa by SDS-PAGE. An isoelectric point of 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 was 45℃, and the enzyme was stable below 35℃. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad β-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl β-glucosides such as p-nitrophenyl β-glucoside, phenyl β-glucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg^<2+>, and reduced by Fe^<3+>, Cu^<2+>, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity ; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl β-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.
社団法人日本農芸化学会の論文
2000-02-23

Purification and Some Properties of a β-Glucosidase from Flavobacterium johnsonae

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