1887

Abstract

Two endoglucanase cDNAs, designated and , were isolated from a cDNA library of the anaerobic fungus . Sequence analysis revealed that 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 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 . 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 β-glucan, lichenin, carob galactomannan,-nitrophenyl β-D-cellobiopyranoside and xylan. Trx-Cel45A′ is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley β-glucan and lichenin.

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2000-08-01
2020-03-31
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