Optimization Of Fermentation Conditions, Purification And Toxicology Analysis Of Chitinase From GS115-pPIC 9k-ChiA 4.0

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) parasitizes on H.armigera, and acts as hydrolyzing the cuticle and peritrophic membranes of insects by chitinase to inhibit their growth and breeding.The gene-engineered strain has been constructed by constructing chitinase gene from HaSNPV into expression vector pPIC9k and transferring into expression host Pichia pastoris GS115. Moreover, multicopy recombinant of GS115-pPIC9k-ChiA was screened and the optimization of fermentation conditions, purification and toxicology analysis of chitinase from GS115-Ppic9k-ChiA4.0 was performed in this study.Methanol utilization plus (Mut⁺) transformant was screened by MM and MD plate culture. The recombinant GS115-pPIC9k-ChiA 4.0 which contradicts 4.0 mg·mL^-1 Geneticin(G418) was obtained with concentration gradient of G418. According to modified Schales, 67.74U·mL^-1 chitinase activity in fermentation culture was obtained after inducing by methanol.Single-factor design and orthogonal test were employed for optimizing cultural conditions. The best cultivating condition was PEP 2%,YNB 0.67%, methanol 1.5%, YE 1.5%, pH 6.0 in inductive medium and adding methanol to 1.5% per 24h, 250rpm, 120 hours inducement in 30℃,in which chitinase activity was 133.70U·mL^-1.The supernatant of fermentation was salted out with (NH₄)₂SO₄. The precipitate was centrifugalized for 25min in 4℃and 13000rpm. The precipitate in 60%⁹0% saturation was collected which was purified with DEAE-Sepharose-FastFlow chromatography which was equilibrated with 25mmol·L^-1 Na₂HPO₄-KH₂PO₄(pH 8.0). Protein peaks were diluted with 0.1M NaCl in 25mmol·L^-1 Na₂HPO₄-KH₂PO₄(pH 8.0). When velocity and sample volumes was 76.4cm·h^-1 and 3.7mL, respectively. 2.795mg chitinase was obtained per 100mL culture and the enzymatic activity was 943.085U·mL^-1. The rate of protein recovery rate and purification fold were respectively 42.7% and 2.78.Based on contact action and stomach toxicity test on Plutella xylostell larvae, the insects death was attributed the hydrolyzation of the cuticle and peritrophic membranes caused by HaSNPV Chitinase. The results of fungicidal test on Bipolaris sorokinianum,Tobacco Alternaria alternate,Tomato botrytis cinerea and Colletitrichum gloeosporiodes indicated that the mechanism of chitinase restrain growth and breeding of fungi was hydrolyzing cell wall. However, it had little effects on Colletitrichum gloeosporiodes.