Aims:  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. Methods and Results:  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. Conclusions:  A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. Significance and Impact of the Study:  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.

Aims:  A new cellobiohydrolase (CBH) gene (cbh3) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris. Methods and Results:  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 °C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper. Conclusions:  A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris. Significance and Impact of the Study:  This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.

Aims:  Microbial proteases play an essential role in scientific research and commercial applications. This study is to clone, sequence, and express a thermostable protease gene from the thermophilic fungi Chaetomium thermophilum and to generate yeast strains expressing C. thermophilum protease suitable for industrial applications. Methods and Results:  Degenerate primers were designed based on the conserved domain of other identified serine proteases and cDNA fragment of C. thermophilum gene pro was obtained through reverse transcriptase‐polymerase chain reaction (RT‐PCR). The full‐length cDNA of 2007 bp was generated using RACE amplification. The cDNA contains an open reading frame of 1596 bp encoding 532 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. The C. thermophilum gene pro was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris, respectively and soluble protein was obtained in P. pastoris. The expressed protease was secreted into the culture medium by the yeast in a functional active form and the purified recombinant protease exhibits optimum catalytic activity at pH 8·0 and 60°C. The enzyme is stable at 60°C. The integration of gene pro into P. pastoris genome is stable after 10 generations and the yeast transformants showed a consistent protease expression. Conclusions:  Gene pro encoding a serine protease from C. thermophilum was cloned, sequenced, and overexpressed successfully in P. pastoris. The expressed protease was purified and the properties of the recombinant protease are characterized. Significance and Impact of the Study:  Chaetomium thermophilum is a soil‐borne thermophilic fungus and the protease cloned from it is stable in a high temperature and a wide rage of pH. The overexpression of the enzyme in a mesophilic micro‐organism offers a potential value for scientific research and commercial applications.