Pastoris Gs115-ppic9k-of Chia 4.0 From Genetically Engineered Bacteria Fermentation Production Hasnpv Chitin Enzyme

Chitinase can decompose chitin specifically. As biological insecticide, chitinase is no harm to animals. Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) is a kind of bollworm-specific pathogenic micro-organism, chitinase as its secretion can inhibit or kill the insects. The laboratory has been studying the recombination, transformation and expression of HaSNPV gene, the goal is to develop non-toxic bio-pesticide.Production of HaSNPV chitinase by gene engineering bacteria GS115-pPIC9k-ChiA 4.0 was studied in the paper. Mainly included optimal condition, scale-up cultivation, separation and purification, as well as the toxicology of HaSNPV chitinase.On the level of shake-flask, fermentation conditions and proportion of main components in induction medium were researched. The optimum conditions for expression of chitinase were as follows:The strain was cultured for 16h-18h in BMGY medium (pH 6.0,30℃, 250rpm), then, resuspended in induction medium (PEP 2%, YNB 1.34%, YE 0.5%, methanol 1%, oleic acid 0.05%, pH 6.0). During the induction phase, adding methanol and glycerol (20:1) per 6h,29℃,250rpm for 120h. Finally, the chitinase concentration was 173.5U/mL.Then,5L fermenter was used for primary scale-up cultivation. Cell concentration should reach about 60g/L before inducted. In the induction phase, keeped DO on 20% to 30% was beneficial to the expression of chitinase. Methanol-Glycerol alternated feeds could further enhance chitinase concentration, the final level was 372U/mL.The supernatant was salted out with (NH4)2SO4 and purified with anion-exchange chromatography.The chitinase concentration was 2885.1U/mL, and the recovery was 69.8%.Based on contact action and stomach toxicity test on Plutella xylostell, the pathological changes were observed by SEM and TEM. Chitin in cell wall was decomposed by HaSNPV chitinase, which caused liquefaction of tissue, resulting in growth retardation or worm's death.HaSNPV chitinase had inhibition on five kinds of common pathogenic fungi, which included Tomato botrytis cinerea, Wheat common rot pathogen, Tobacco brown spot pathogen, Tomato alternaria solani and Colletitrichum gloeosporiodes.