Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, ce/B29 and ce/B2?, were isolated from a cDNA library d erived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii s train SG4. The nucleotide sequences of celB2 and celB29 and the primary str uctures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966 bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54 kDa. Analysis of the 1451 bp celB2 cDNA revealed a n 1164 bp open reading frame coding for a 44 kDa protein consisting of 388 amino acids, Both deduced proteins:had a high sequence similarity in centra l regions containing putative catalytic-domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hyd rolase family 5 and were most closely related to endoglucanases from the an aerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpi nomyces sp. The classification of CelB29 and CelB2 as endoglucanases was su pported by enzyme assays. The cloned enzymes had high activities towards ba rley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel , laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 sh owed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G2) to p-nitro phenyl-P-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyr anoside (pNP-G(1),) with preferential activity against p-nitrophenyl-beta-D -cellotrioside (pNP-G(3),). Based on these results, we proposed that CelB29 and CelB(2) are endoglucanases with broad substrate specificities for shor t- and long-chain beta-1,4-glucans. (C) 2000 Elsevier Science B.V, All righ ts reserved.