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Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equi

Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK¹
Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK²

Author for correspondence: Ruth Y. Eberhardt. Tel: +44 1223 496478. Fax: +44 1223 496023. e-mail: ruth.eberhardt{at}bbsrc.ac.uk

Two endoglucanase cDNAs, designated cel5A and cel45A, were isolated from a cDNA library of the anaerobic fungus Piromyces equi. Sequence analysis revealed that cel5A has an open reading frame of 5142 bp and encodes a 1714 amino acid modular enzyme, Cel5A, with a molecular mass of 194847 Da. Cel5A consists of four catalytic domains homologous to family-5 glycosyl hydrolases, two C-terminal dockerins and one N-terminal dockerin. This is the first report of a complete gene containing tandem repeats of family-5 catalytic domains. The cDNA cel45A has an open reading frame of 1233 bp and encodes a 410 amino acid modular enzyme, Cel45A, with a molecular mass of 44380 Da. The catalytic domain, located at the C terminus, is homologous to the family-45 glycosyl hydrolases. Cel45A is the first family-45 enzyme to be described in an anaerobe. The presence of dockerins at the N and C termini of Cel5A and at the N terminus of Cel45A implies that both enzymes are part of the high-molecular-mass cellulose-degrading complex produced by Piromyces equi. The catalytic domain nearest the C terminus of Cel5A and the catalytic domain of Cel45A were hyperexpressed as thioredoxin fusion proteins, Trx-Cel5A' and Trx-Cel45A', and subjected to biochemical analysis. Trx-Cel5A' has a broad substrate range, showing activity against carboxymethylcellulose, acid-swollen cellulose, barley ß-glucan, lichenin, carob galactomannan,p-nitrophenyl ß-D-cellobiopyranoside and xylan. Trx-Cel45A' is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley ß-glucan and lichenin.

Keywords: anaerobic fungi, cellulase, glycosyl hydrolases (families 5 and 45), endoglucanases, Piromyces equi

Abbreviations: CMC, carboxymethylcellulose; CMCase, carboxymethylcellulase; DNSA, dinitrosalicylic acid reagent; pNPC, p-nitrophenyl ß-D-cellobiopyranoside

The EMBL accession numbers for the sequences reported in this paper are AJ277482 and AJ277483.

^a Present address: Finnfeeds International, PO Box 777, Marlborough SN8 1XN, UK.

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