KMID : 0880220080460010051
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Journal of Microbiology 2008 Volume.46 No. 1 p.51 ~ p.55
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Purification and Characterization of Thermostable ¥â-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris Grown on Microcrystalline Cellulose
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Yoon Jeong-Jun
Kim Ki-Yeon Cha Chang-Jun
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Abstract
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An extracellular ¥â-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC- MS/MS suggested that the protein has high homology with fungal ¥â-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-¥â-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified ¥â-glucosidase was observed at pH 4.5 and 70¡ÆC. The F. palustris protein exhibited half-lives of 97 h at 55¡ÆC and 15 h at 65¡ÆC, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-¥â-lactoside, p-nitrophenyl-¥â-xyloside, p-nitrophenyl-¥á-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the ¥â-glucosidase from F. palustris can be classified as an aryl-¥â-glucosidase with cellobiase activity.
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KEYWORD
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¥â-glucosidase, brown-rot fungus, Fomitopsis palustris, purification, microcrystalline cellulose
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