The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a β-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular β-glucosidase activity. To identify the component in the fungal cellulosome responsible for the β-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737bp 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 β-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent Km and Ki values of heterologous Cel3A and the fungal cellulosome for p-nitrophenyl-β-d-glucopyranoside and d-glucono-1,5-Δ-lactone respectively indicated that cel3A encodes the β-glucosidase activity of the Piromyces sp. strain E2 cellulosome.
Abbreviations used: GH, glycoside hydrolase; ORF, open reading frame; pNP, p-nitrophenyl.
The nucleotide sequence data reported is available in the GenBank® Nucleotide Sequence Database under the accession number AY172977.