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Beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase.

ABSTRACT: The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.

SUBMITTER: Steenbakkers PJ

PROVIDER: S-EPMC1223235 | biostudies-other | 2003 Mar

REPOSITORIES: biostudies-other

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Project description:Background. The anaerobic gut fungi (phylum Neocallimastigomycota) represent a promising source of novel lignocellulolytic enzymes. Here, we report on the cloning, expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme (Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomycessp. strain C1A under different growth conditions. This represents the first study of a GH39-family enzyme from the anaerobic fungi. Methods. Using enzyme activity assays, we performed a biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH and temperature values to identify the optimal enzyme conditions and the specificity of the enzyme. In addition, substrate competition studies and comparative modeling efforts were completed. Results. Contrary to the narrow range of activities (β-xylosidase or α-L-iduronidase) observed in previously characterized GH39 enzymes, Bgxg1 is unique in that it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase activities (11.5 ± 1.2, 73.4 ± 7.15, and 54.6 ± 2.26 U/mg, respectively) and a weak xylanase activity (10.8 ± 1.25 U/mg), as compared to previously characterized enzymes. Further, Bgxg1 possesses extremely high affinity (as evident by the lowest K m values), compared to all previously characterized β-glucosidases, β-galactosidases, and xylanases. Physiological characterization revealed that Bgxg1 is active over a wide range of pH (3-8, optimum 6) and temperatures (25-60 °C, optimum 39 °C), and possesses excellent temperature and thermal stability. Substrate competition assays suggest that all observed activities occur at a single active site. Using comparative modeling and bioinformatics approaches, we putatively identified ten amino acid differences between Bgxg1 and previously biochemically characterized GH39 β-xylosidases that we speculate could impact active site architecture, size, charge, and/or polarity. Discussion. Collectively, the unique capabilities and multi-functionality of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass.

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Beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase.

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