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

Lookup NU author(s): Emeritus Professor Harry Gilbert

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Abstract

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. Ce145A 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 Ce145A 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 Ce145A 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 beta-glucan, lichenin, carob galactomannan, p-nitrophenyl beta-D-cellobiopyranoside and xylan, Trx-Cel45A' is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley beta-glucan and lichenin.


Publication metadata

Author(s): Eberhardt RY, Gilbert HJ, Hazlewood GP

Publication type: Article

Publication status: Published

Journal: Microbiology

Year: 2000

Volume: 146

Pages: 1999-2008

ISSN (print): 1350-0872

ISSN (electronic): 1465-2080

Publisher: Society for General Microbiology

URL: http://mic.sgmjournals.org/content/146/8/1999.full.pdf


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