Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Identification and Functional Analysis of a Gene Encoding β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris: Hwang-Woo Ji , Chang-Jun Cha; J. Microbiol. 2010;48(6):808-813. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0482-2

Abstract: The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.

Purification and Characterization of Thermostable β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris Grown on Microcrystalline Cellulose: Jeong-Jun Yoon , Ki-Yeon Kim , Chang-Jun Cha; J. Microbiol. 2008;46(1):51-55.; DOI: https://doi.org/10.1007/s12275-007-0230-4

Abstract: An extracellular β-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 β-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-β-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 β-glucosidase was observed at pH 4.5 and 70°C. The F. palustris protein exhibited half-lives of 97 h at 55°C and 15 h at 65°C, 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-β-lactoside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the β-glucosidase from F. palustris can be classified as an aryl-β-glucosidase with cellobiase activity.

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