Microencapsulated Genetic Engineering Yeast Metabolism Production Chitinase And Its Control Effect On Lepidoptera Larvae

Chitinase had became one of the most promising hotspots about biological pesticides based on its specific hydrolysis function. Gene engineering strain (GS115-pPIC9k-Chi A4.0) which constructed by gene recombination technology could produce chitinase in the induction stage. However, the production of chitinase isn’t high in the free culture, it is caused by the high mechanical shear and product inhibition. In order to improve this shortcoming, microencapsulation was used in this paper. Accordingly, Sodium alginate and chitosan were used as material to prepare microcapsule, and the microencapsulated cell was gene engineering strain GS115-pPIC9k-ChiA4.0. It carried out a study to optimize conditions for chitinase production, evaluate the feasibility of microencapsulated gene engineering strain, and research the control effect of its microencapsulated metabolites on lepidoptera larvae. Experimenta sesults were should as follow:l.The cells number and the chitinase production reached the maximum value in the optimal contition of cells initial inoculum OD6oo=0.339(1mL),1%methanol addition,72h growth time and55h induce time.2.The dry weight loss of100KD and200KD chitosan membrane were16.39%and10.24%after the degradation of96h in the fermentation liquid of gene engineering strain.3. Infrared analysis showed poly electrolyte (PEC) composite film was generated by ionic electrostatic interaction between chitosan and alginate gel, the damage rate of microcapsules was14.67%in the500U/mL chitinase solution after120h. Therefor, AC microcapsule was stable for chitinase. When the microcapsules loaded chitinase was placed in two PBS buffer(pH6.0and7.8) after96h, the release rate of chitinase are32.21%and74.92%respectively.4.Compared to the free cultrue, microencapsulation could get greater cells number (OD600=1.811) and chitinase production (601.8U/mL, it higher than free culture39.3U/mL). Moreover, microencapsulation also extended stable period of growth of gene engineering strain. Finally, the morphologies of microcapsule nearly remained intact in the whole process of growth and metabolism. 5.500U/mL metabolites of microencapsulated gene engineering strain didn’t have sig-nificant antifeedant effect for plutella xylostella larvae, which was detected by leaf dipping method. Namely, the maximal value of selective and non-selective antifeedant rate were6.23%and11.41%respectively.6.The metabolites could control larvae by two methods involved leaf dipping method and drop method. Different concentrations of metabolites had different insecticidal effect, the indoor toxic effect of3000U/mL was most obvious for the plutella xylostella larvae among six groups of concentration (100U/mL、250U/mL.500U/mL、1000U/mL、2000U/mL3000U/mL), which corrected mortality of leaf dipping method and drop method were72.22%,82.22%. Two treatment methods had inhibition on the larvae feeding and growth, and inhibitory effect of drop method higher than leaf dipping method, what’s more, inhibitory effect also increased along with the increase of metabolite concentration. In addition, insecticidal aging of micro-encapsulated cells metabolites was longer than chitinase for2-3d.7.Six concentrations of microencapsulated cells metabolite had different effects on pupation and emergence, delayed mortality increased along with the increase of the concetration.3000U/mL metabolite had a serious long-term delayed mortality on larvae, which involved pupation and eclosion. The pupation and eclosion rate of larvae detected by stomach poison and contact were5.56%,0%and1.93%,0%, respectively.Therefor,the alginate/chitosan microcapsule immobilized gene engineering strain GS115-pPIC9k-ChiA4.0was an effective tool for biological pesticides chitinase production. The metabolites of microencapsulated cells had the function of biological degradation for insects, it could be used for pest control.