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

Two endoglucanase cDNAs, designated cel5A and cel45A, were isolated from a cDNA library of the anaerobic fungus Piromyces equi. Sequence analysis reve aled that cel5A has an open reading frame of 5142 bp and encodes a 1714 ami no acid modular enzyme, Cel5A, with a molecular mass of 194847 Da. Cel5A co nsists of four catalytic domains homologous to family-5 glycosyl hydrolases , two C-terminal dockerins and one N-terminal dockerin. This is the first r eport of a complete gene containing tandem repeats of family-5 catalytic do mains. The cDNA cel45A has an open reading frame of 1233 bp and encodes a 4 10 amino acid modular enzyme, Cel45A, with a molecular mass of 44380 Da. Th e catalytic domain, located at the C terminus, is homologous to the family- 45 glycosyl hydrolases. Ce145A is the first family-45 enzyme to be describe d 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 dom ain 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, aci d-swollen cellulose, barley beta-glucan, lichenin, carob galactomannan, p-n itrophenyl beta-D-cellobiopyranoside and xylan, Trx-Cel45A' is active again st carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glu cans, barley beta-glucan and lichenin.