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Genetics and Molecular Biology |
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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