Molecular Cloning and Expression of a Family 7 Cellobiohydrolase Gene Cbh1 From Penicillium Funiculosum Ncl1

Abstract

A cellobiohydrolase gene cbh1 was cloned from a cellulolytic fungus, Penicillum funiculosum NCL1. Nucleotide sequencing of cbh1 gene revealed that this gene was 1590 bp length encoding a putative protein consisting of 529 amino acids. The deduced amino acid sequence showed that the predicted molecular mass of the CBHI was 54.9 kDa, and showed significant homology to glycoside hydrolase family 7 cellobiohydrolases. The cbh1 gene was cloned using pET30b and expressed in E. coli BL21 (DE3). The expression analysis of the recombinant E. coli BL21 (pETC7) revealed the production of cbh1 transcript; however, functional cellobiohydrolase could not be detected. Therefore, the cbh1 gene was sub-cloned into GST tagged expression vector pGEX4t-3. The GST tagged cellobiohydrolase was purified to homogeneity using affinity chromatography. The recombinant enzyme exhibited optimum catalytic activity at pH 5.0 and 50 °C respectively. It was thermostable at 50 °C and retained 70% of its original activity after 30 min at 60 °C.