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Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris
- Chen, J., Zhang, Y.-Q., Zhao, C.-Q., Li, A.-N., Zhou, Q.-X., Li, D.-C.
- Journal of applied microbiology 2007 v.103 no.6 pp. 2277-2284
- Chaetomium, Pichia pastoris, RNA, active sites, alcohols, amino acids, catalytic activity, complementary DNA, culture media, enzyme activity, enzyme stability, gene overexpression, genes, glucan 1,4-alpha-glucosidase, glycoproteins, half life, molecular biology, molecular cloning, open reading frames, pH, reverse transcriptase polymerase chain reaction, saccharification, signal peptide, starch, thermal stability, thermophilic fungi, yeasts
- Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4·5-5·0 and 65°C. The enzyme was stable at 60°C, the enzyme activity kept 80% after 60 min incubation at 70°C. The half-life was 40 and 10 min under incubation at 80 and 90°C respectively. A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.