title: Improvement of chitinase Pachi with nematicidal activities by random mutagenesis.
abstract: Chitinase, an enzyme that can degrade the main compositions of insect intestine and cuticle, has been used in the bio-control field. Our previous work has reported the chitinase Pachi with nematicidal activity (Caenorhabditis elegans). In the present study, to improve the chitinolytic and nematicidal activities of Pachi, a random mutant library was constructed by error-prone PCR and screened by bacteriophage T7-based high-throughput screening system. One mutant, PachiN35Dwas obtained from about 10, 000 clones. The kinetics analysis revealed that PachiN35Dexhibited a 63% decrease in Kmvalue against chitosan, a 2.1-fold enhancement in kcat/Kmvalue and a 1.2-fold increase in specific activity over the wild-type Pachi. Moreover, the mortality analysis against Caenorhabditis elegans showed that the 50% lethal concentration (LC50) of PachiN35Dis 309.6±1.1μg/ml and a 20% increase in nematicidal activity over the wild-type Pachi (with a LC50value of 387.3±31.7μg/ml). The structure modeling and superimposition indicated that the substitution N35D reduced the distance between substrate and substrate-binding site Asp141, finally resulting in an increase in substrate affinity, catalytic efficiency and specific activity. These results provide useful information for the study of structure-function relationship of Pachi and lay a foundation for its potential applications in agro-biotechnology.
authors.affiliation: College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China.
authors.forename: Junpeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China.
authors.forename: Yangdongfang
authors.initials: Y
authors.lastname: An
authors.affiliation: College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D000969:Antinematodal Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017173:Caenorhabditis elegans / Q000187:drug effects; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D013696:Temperature
chemical_list: D000969:Antinematodal Agents; D008670:Metals; D002688:Chitinases
keywords: Caenorhabditis elegans; Chitinase; Nematicidal; Pseudomonas aeruginosa; Random mutant library