Biochemical Characterization Of The Chitin Deacetylase BmCDA1

Chitin,the ?-(1,4)-linked homopolymer of N-acetylglucosamine,is one of the major components of insect exoskeleton.The insect chitin deacetylase 1(CDA1)is a potential pesticide target which can catalyse the deacetylation of chitin during molting.The RNA interference of CDA1 would cause abnormal molting and death.However,the knowledge about the biochemical properties of this enzyme is very limited.This work focused on the recombinant expression and crystal condition of BmCDA1-CAD(catalytic domain),and studied the substrate binding activity,catalytic activity.Besides,the dsRNA of Ostrinia furnacalis CDA1 was synthesized in Escherichia coli for application in pest control.The work in this thesis includes:(1)The expression vector of pPIC9-BmCDA1-CAD was constructed and introduced into Pichia pastoris.The recombinant proteins of BmCDA1-CAD were enriched through 75% ammonium sulfate precipitation from the fermentation,purified through metal-chelating affinity chromatography and verified by SDS-PAGE and Western Blot.(2)Initial crystallization screening was carried out in 48-well tissue-culture plates at 277 K by the hanging-drop vapor diffusion method using commercially available kit containing 400 conditions.The protein buffer is 20 mM Bis-Tris,10 mM NaCl,pH 7.0.Initial crystallization screening yielded a crystallization hit under the condition of 0.2 M Ammonium sulfate,0.1 M BIS-TRIS pH 6.5,25% w/v Polyethylene glycol 3,350.This condition was subsequently optimized by testing 363 new crystallization combinations with varying protein concentrations,pH values and Polyethylene glycol 3,350 concentrations.Finally,we obtained an optimal composition for the reservoir solution of 0.2 M Ammonium sulfate,0.1 M BIS-TRIS pH 6.8,27% w/v Polyethylene glycol 3,350.Protein concentration was 12 mg/ml.(3)Compared with the binding abilities of negative control BSA(12%-15%)to four substrates,the chitin binding assay indicated that the binding abilities of BmCDA1(40%)to ?-chitin was stronger than BmCDA1-CAD(33%);the binding abilities of BmCDA1(53%)to ?-chitin was stronger than BmCDA1-CAD(32%);the BmCDA1(27%)could bind colloidal chitin,but BmCDA1-CAD(17%)couldn't;both BmCDA1(16%)and BmCDA1-CAD(12%)couldn't bind chitosan.(4)The research team have proved that the catalytic activities of BmCDA1 and BmCDA7 was undetectable with the three known methods by detecting free amino groups.So,we established a new method by detecting free acetic acid.When using 65% acetylated chitosan as substrate,BmCDA7 showed deacetylation activity using the microcrystalline acetic acid test kit.It illustrated that the new method was workable.But BmCDA1 and BmCDA1-CAD showed no activity.The structure analysis by homologous modeling showed a longer LOOP covering the catalytic pocket of BmCDA1-CAD compared with BmCDA7.The expression vector of pPIC9-BmCAD1/7 was constructed and introduced into Pichia pastoris.The recombinant protein BmCAD1/7 was enriched through 75% ammonium sulfate precipitation from the fermentation.(6)The genes for expressing dsRNA for OfCDA1,Of EcR and GFP were cloned into the vector L4440 respectively and transformed into E.coli cells HT115.The dsRNAs were expressed and extracted from the recombinant cells at a yield of 3 g/L,0.15 g/L and 0.12 g/L respectively.The best concentration of bacterium for induction was OD600 =0.3.The extracted dsRNAs were stable below 65?,and were resistant to RNase A and DNase 1 treatment.This work will help to study the genes function through oral delivery of ds RNAs,and help to control the insect pest Ostrinia furnacalis.