Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungusChaetomium thermophilumand its expression inPichia pastoris

Abstract

A new cellobiohydrolase (CBH) gene (cbh3) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris. Using RACE-PCR, a new thermostable CBH gene (cbh3) was cloned from C. thermophilum. The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilum CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris. The recombinant CBH purified was a glycoprotein with a size of about 48.0 kDa, and exhibited optimum catalytic activity at pH 5.0 and 60 degrees C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper. A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris. This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.