β-D-Glucosidase from Seeds of Japanese Cycad, Cycas revoluta

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Abstract
β-D-Glucosidase was purified from seeds of Japanese cycad by dialysis, chromatography on CM-Sepharose CL-6B, gel filtration on Biogel P-200, and chromatofocusing. By chromatofocusing, β-D-glucosidase was separated into four components whose isoelectric points were in a very narrow range (7.43-7.68). All these components were glycoproteins. The main component (pI =7.59) was homogeneous on gel isoelectric focusing, and was crystallized from ammonium sulfate solution. The molecular weight of the crystalline preparation was determined to be 137,000 by gel filtration, and 67,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating the main component was composed of two subunits with the same molecular weight. The amino acid composition and sugar content of the main component were also determined. All four components hydrolyzed not only o-nitrophenyl β-D-glucopyranoside but also o-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-fucopyranoside, and o-nitrophenyl β-D-xylopyranoside. Hydrolysis rates of each substrate by the four components were quite similar. Mixed substrate experiments using crystalline preparation proved that a single active site was responsible for the hydrolysis of these substrates.