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Volume 146, Issue 8

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

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

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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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Keyword(s): anaerobic fungi , cellulase , CMC, carboxymethylcellulose , CMCase, carboxymethylcellulase , DNSA, dinitrosalicylic acid reagent , endoglucanases , glycosyl hydrolases (families 5 and 45) , Piromyces equi and pNPC, p-nitrophenyl β-D-cellobiopyranoside
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Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equiThe EMBL accession numbers for the sequences reported in this paper are AJ277482 and AJ277483.
Microbiology 146, 1999 (2000); https://doi.org/10.1099/00221287-146-8-1999
/content/journal/micro/10.1099/00221287-146-8-1999
/content/journal/micro/10.1099/00221287-146-8-1999
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