Mining and characterization of two novel chitinases from Hirsutella sinensis using an efficient transcriptome-mining approach

Abstract

Two novel family 18 chitinases, chiA and chiH, were identified and cloned from the transcriptome of H. sinensis based on the transcriptome sequence data. The recombinant chitinases were overexpressed in Escherichia coli BL21, subsequently purified and functionally characterized. The optimal temperature and pH for chiA were 55 °C and 5.0, respectively, and those for chiH were 50 °C and 5.0, respectively. The highest enzyme activities of 11.5 U/mg and 8.1 U/mg were obtained for chiA and chiH, respectively, when colloidal chitin was used as the substrate with Ba2+. chiA exhibited higher Vmax of 1.94 μmol/μg/h and kcat of 1.443 S−1 than those of chiH with Vmax of 1.63 μmol/μg/h and kcat of 1.175 S−1, and both were efficient towards colloidal chitin compared with other typical family 18 chitinases. Substrate specificity and gene expression analyses indicated that chiA and chiH preferred substrates containing N-acetyl groups, such as colloidal chitin and glycol chitin, while no activity was detected toward laminarin, cellobiose, carboxymethyl cellulose and starch. The work presented here would aid in the understanding and performance of future studies on the infection mechanism of H. sinensis.