Duochuan Li

Two novel thermostable chitinase genes from thermophilic fungi: cloning, expression and characterization.

Two chitinase genes, Tachit1 from Thermoascus aurantiacus var. levisporus and Ctchit1 from Chaetomium thermophilum were isolated. Tachit1 and Ctchit1 encode putative single-domain proteins (TaCHIT1 and CtCHIT1) of 399 and 402 amino acid residues, respectively. The catalytic domains of TaCHIT1 and CtCHIT1 are similar to those of other fungal chitinases in family 18 of glycosyl hydrolases. TaCHIT1 and CtCHIT1 have a molecular weight of about 48.4 and 47.3kDa, respectively when produced in recombinant Pichia pastoris. The enzymes exhibited optimum catalytic activity at pH 8.0 and 50 degrees C for TaCHIT1 and at pH 5.5 and 60 degrees C for CtCHIT1. TaCHIT1 retained 95.3% of its activity after 60 min at 50 degrees C. CtCHIT1 was stable at 50 degrees C and retained 96.7% of its activity after 60 min incubation at 60 degrees C. The TaCHIT1 and CtCHIT1 produced Glc-NAc2 as the major product, when colloidal chitin was used as the substrate. The enzyme could not hydrolyze pNp-(GlcNAc), but hydrolyzed colloidal chitin, powdery chitin and chitosan. These features make these proteins potentially useful for applications requiring chitin hydrolysis at elevated temperatures.

Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris

Aims:  A new cellobiohydrolase (CBH) gene (cbh3) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris.

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on deae-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 μg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. n-terminal amino acid sequencing (10 residues) was kx (x is uncertain) tlpdlkyd. The n-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

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.

Cloning, expression and characterization of the serine protease gene from Chaetomium thermophilum

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.

Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.