title: 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 Vmaxof 1.94 μmol/μg/h and kcatof 1.443 S-1than those of chiH with Vmaxof 1.63 μmol/μg/h and kcatof 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.
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Shan
authors.initials: S
authors.lastname: Lin
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Ming
authors.initials: M
authors.lastname: Yi
authors.affiliation: East China Pharmaceutical Group Limited Co., Ltd, Hangzhou 311000, Zhejiang, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.affiliation: East China Pharmaceutical Group Limited Co., Ltd, Hangzhou 311000, Zhejiang, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D001203:Ascomycota / Q000201:enzymology / Q000235:genetics; D002688:Chitinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D059467:Transcriptome
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002688:Chitinases
keywords: Chitinase; Gene expression; Hirsutella sinensis; Overexpression; Substrate specificity