Cloning of an exo-β-1,4-D-glucanases Gene(cbh1) from Penicillium oxalicum, Its Expression in Pichia pastoris and Characterization of Recombination CBHⅠ
Abstract：Most of the Trichoderma strains are deficient in the β-glucosidase production, results in end product inhibition, and the cellulase from Trichoderma shows relatively low growth rates, Thus, cellulase from Penicillium has attracted renewed attention. The complete gene of exo-β-1,4-D-glucanases (cbh1) was cloned and expressed in Pichia pastoris to characterize the recombinant enzyme. The gene of cbh1 (GenBank Accession No.HQ843504), cloned from M strain of Penicillium oxalicum by the modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was 1 641 bp without intron and encoding 547 amino acids. Because its Glu236, Asp238 and Glu241 were the classical catalytic residues, we predicted cbh1 belongs to glycoside hydrolase family 7. The recombinant plasmid pPIC9-cbh1 was constructed by cloning the gene into vector pPIC9 and then was transformed into Pichia pastoris strain GS115 by electroporation. The sequencing and activity analysis of the positive recombinant showed pPIC9-cbh1 could successfully express cbh1 in P. pastoris. To our knowledge, it was the first report that exo-β-1,4-D-glucanases from Penicillium oxalicum successfully expressed in P. pastoris. The enzyme activity of the recombinant reached up to 56 IU/mg. The optimum pH and temperature of the recombinant enzyme were 6.0 and 55℃ respectively, and the stability of pH and temperature were both good. The results suggest that cloning of the cbh1 enriches the gene resources of cellulases, and its successful expression in P. pastoris provides the fundament for the construction of gene engineering strains.
