title: 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.
authors.affiliation: Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Steenbakkers
authors.forename: Harry R
authors.initials: HR
authors.lastname: Harhangi
authors.forename: Mirjam W
authors.initials: MW
authors.lastname: Bosscher
authors.forename: Marlous M C
authors.initials: MM
authors.lastname: van der Hooft
authors.forename: Jan T
authors.initials: JT
authors.lastname: Keltjens
authors.forename: Chris
authors.initials: C
authors.lastname: van der Drift
authors.forename: Godfried D
authors.initials: GD
authors.lastname: Vogels
authors.forename: Huub J M
authors.initials: HJ
authors.lastname: op den Camp
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002475:Cellobiose / Q000737:chemistry / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D020089:Piromyces / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D001617:beta-Glucosidase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D002475:Cellobiose; D002482:Cellulose; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase