Preparation Of Nanochitinases And Synergistic Insecticidal Effect On Bacillus Thuringiensis Preparations

Bacillus thuringiensis (Bt) is a spore-forming bacterium that has the ability to synthesize a large amount of insecticidal crystal proteins (ICPs) during growth and metabolism, which leads to develop the most important bacterial insecticide for controlling the target pests at the global scale. Various Bt-derived pesticides have proven that they are relatively highly insect-specific, remarkably safe for humans, other mammals, and non-target fauna, and cost-effective for production in general, however, during the long-term practice of applying Bt pesticides, it is also recognized some of their drawbacks in terms of delayed toxicity, weak resistant capability to harsh environmental factors especially UV irradiation and high temperature, and relatively short persistence in the field. This study focuses on the synergistic insecticidal effects of various chitinases including the nanochitinases, the mutated chitinase with high chitinolytic activity and surface-displayed chitinases, on the larvae of Caenorhabditis elegans and Helicoverpa armigera, by preparing several nanochitinases, expressing and purifying a mutated chitinase, and constructing the recombinant strains displaying the chitinases onto surface of Bt cells.The surface groups of a hydroxylated nano SiO2material were initially chemically modified through amination, carboxylation and hydroformylation to prepare aminated nano SiO2, carloxylated nano SiO2and hydroformylated nano SiO2, respectively. Following SEM examination, particle diameter measurement and Fourier transform infrared spectrometer analysis of the prepared nano SiO2materials, the composite chitinases immobilized to various nano SiO2materials through either covalent or statically adsorbed linkages, namely, the "Nano SiO2-CH=N-Chi9602" through covalent linkage; the "Nano SiO2-CONH-Chi9602" through covalent linkage; the "Nano SiO2-COOH/Chi9602","Nano SiO2-NH2/Chi9602", and "Nano SiO2-OH/Chi9602" all through statically adsorbed linkages, were obtained. The immobilization rate and specific chitinase activity were then comparatively determined. It showed that the variant immobilization rate by90%,87.7%,29.5%,80.2%, and55.8%, and also variant specific enzyme activity by3%,12,8%,93.8%,94%and44.8%compared to the original chitinase Chi9602, of Nano SiO2-CH=N-Chi9602, Nano SiO2-CONH-Chi9602, Nano SiO2-COOH/Chi9602, Nano SiO2-NH2/Chi9602, and Nano SiO2-OH/Chi9602, respectively. Thus, because of relatively high immobilization rate and specific enzyme activity, the Nano SiO2-NH2/Chi9602was chosen as optimized nanochitinase for further usage in this study.The stability under different temperature and pH ranges of Nano SiO2-NH2/Chi9602were investigated. It showed that relatively high stability in terms of higher temperature and the wider pH in compare with the free Chi9602. Further, the stability under UV irradiation was comparatively evaluated. The half-life of Nano SiO2-NH2/Chi9602enzymatic activity was23.4min under given experimental condition, whereas the half-life of Chi9602enzymatic activity was15.1under similar condition, indicated that Nano SiO2-NH2/Chi9602exhibited more high stability towards UV irradiation.Bioassays against the larvae of C. elegans using the mixtures with Nano SiO2-NH2/Chi9602and YBT-020insecticidal preparations were performed. It showed a remarkable synergistic insecticidal effect by adding Nano SiO2-NH2/Chi9602compared to alone YBT-020preparation. Similar results were obtained in the parallel experiment of the other insecticidal Bt bacterium, YBT-032. Although no obvious increase of synergistic effect in compare with the free Chi9602, however, Nano SiO2-NH2/Chi9602exhibited more rapid lethality against the larvae of C. elegans. The more loading and the wider distribution of Nano SiO2-NH2/Chi9602inside the larvae bodies appeared to support this property of Nano SiO2-NH2/Chi9602.The synergistic insecticidal effect of a mutated chitinase, Chi9602W50A, on the larvae of C. elegans was investigated by adding the purified enzyme into the insecticidal preparations of YBT-020. It showed apparently increased lethality of Chi9602W50A over that of Chi9602. Interestingly, when added Chi9602W50A into the preparations of YBT-1520that has been proven previously the high toxicity against the larvae of Helicoverpa armigera, the mixtures exhibited an LC50of4.238μg·mL-1, a23.8%decrease over the mixtures with Chi9602and the preparations of YBT-1520, indicated the remarkable synergistic insecticidal activity against the larvae of H, armigera.Based on above results, a recombinant Bt strain MB461was constructed for displaying the Chi9602W50A onto surface of Bt recipient strain YBT-881, aims at obtaining an engineered Bt strain with synergistic insecticidal activity under the action of self-displayed chitinases. Unfortunately, neither alone insecticidal activity of MB461as well as YBT-881, nor the synergistic effect was found from the bioassays against the larvae of C. elegans, suggested that chitinase could only endow the synergistic insecticidal activity but not a newly-generated insecticidal activity. This conclusion was further supported by the bioassays of the mixed insecticidal preparations of both MB461 and YBT-020.