Cloning, expression, and characterization of two novel cuticle-degrading serine proteases from the entomopathogenic fungus Cordyceps sinensis

The entomopathogenic fungus Cordyceps sinensis has been important in traditional Chinese medicine, but is yet to be commercially cultivated. Difficulty in cultivation results in part from the low percentage of fungal infection on artificially inoculated host insects. To better understand the infection mechanism, we cloned two cuticle-degrading serine protease genes (csp1 and csp2) from C. sinensis. These enzymes are novel members of the S8A subfamily of proteases. Identities of cDNA or amino acid sequences between Csp1 and Csp2 were 72.9% and 68.9%, respectively. After successful expression in the yeast Pichia pastoris, recombinant enzymes were purified and characterized using the synthetic substrate N-suc-AAPF-p-NA. Both were chymotrypsin-like serine proteases with an optimum pH of 7.0 and an optimal temperature of 40 °C (Csp1) or 50 °C (Csp2). Bioassay revealed that Csp1 and Csp2 degraded the cuticle proteins of larval Hepialus sp. in vitro. This is the first report of serine proteases from C. sinensis.