Isolation of the gene and characterization of the enzymatic properties of a major exoglucanase of humicola grisea without a cellulose-binding domain.

Abstract

An exoglucanase gene was cloned from a cellulolytic fungus, Humicola grisea. DNA sequencing of this gene, designated as exo1, revealed that it contained four introns in the coding region. The deduced amino acid sequence of EXO1 was 451 amino acids in length and showed 57.7% identity with that of H. grisea cellobiohydrolase 1 (CBH1), but lacked the typical domain structures of a cellulose-binding domain and a hinge region. Transcriptional analysis of the exo1 and cbh1 genes showed that the expression of these genes was induced by Avicel, and repressed in the presence of glucose. The exo1 gene was expressed in Aspergillus oryzae, and the recombinant EXO1 protein was purified. EXO1 and CBH1 produced by A. oryzae showed relatively higher activity toward Avicel, but showed much lower activity toward carboxymethyl cellulose (CMC) and p-nitrophenyl-beta-D-cellobioside (PNPC), than H. grisea endoglucanase 1 (EGL1). The addition of a cellulose-binding domain and a hinge region to EXO1 caused decreases in its enzymatic activities as well as the deletion of the cellulose-binding domain from CBH1. EXO1 showed relatively weak or no synergistic activity toward Avicel with H. grisea endoglucanases, but showed a significant level of apparent synergism with H. grisea CBH1 and Trichoderma reesei EGLI. CBH1 showed a significant level of apparent endo-exo synergism with H. grisea and T. reesei endoglucanases. H. grisea has at least two different types of major exoglucanase components and shows strong cellulolytic activity through synergism with cellulase components including EXO1 and CBH1.