Improvement of chitinase Pachi with nematicidal activities by random mutagenesis

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, Pachi_N35D was obtained from about 10, 000 clones. The kinetics analysis revealed that Pachi_N35D exhibited a 63% decrease in K_m value against chitosan, a 2.1-fold enhancement in k_cat/K_m value 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 Pachi_N35D is 309.6±1.1μg/ml and a 20% increase in nematicidal activity over the wild-type Pachi (with a LC₅₀ value 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.