Molecular Cloning And Biochemical Characterization Of Group ? Chitinase From The Insect Ostrinia Furnacalis

Chitin, the main component of the cuticle, is vital for insect survival. Chitin has to be regularly synthesized and degraded to meet the requirement of insect growth and development. Chitinase is a key enzyme in the process of chitin metabolism. Insect chitinases can be divided into ten groups and group ? chitinase has an N-terminal transmembrane segment and a special domain composition with two catalytic domains (CADs) and one chitin binding domain (CBD). Because the physiological importance of group ? chitinase has been proved by RNA interference, this enzyme is a potential target for green pesticide design. However, because group ? chitinase is not found in the molting fluid, its exact physiological is unknown. Here, cloning, expression and characterization of OfCht?, the group ? chitinase from Ostrinia furnacalis, an important agricultural pest, were performed to provide information for revealing its physiological function.The work in this thesis includes:(1) The gene of OfCht? from insect O, furnacalis was obtained through PCR and RACE. The full length of OfCht? was 4193-bp and the coding length was 2961-bp encoding 987 amino acids. The N-terminal carried a transmembrane domain with 23 amino acids. OfCht? had two catalytic domains (CADs) and one chitin binding domain (CBD) and phylogeneic analysis demonstrated that OfCht? belongs to group ? chitinases.(2) The two CADs of OfCht?-1, OfCht?-2/OfCht?-?2 were successfully expressed in the Pichia pastoris GS115 cells. Recomibinant OfCht?-2 and OfCht?-?2 were purified by ammonium sulfate precipitation and chelating affinity chromatography and OfCht?-1 was purified by cation-exchange chromatography. The mass spectrometry results showed that the cleavage of OfCht? occurred in the sites of Arg503 and Lys510, resulting the loss of "VKKPTKK".(3) Characterization of enzymatic properties showed that OfCht?-1, OfCht?-2 and OfCht?-?2 had the same optimum pH 6.0. OfCht?-1 had higher affinity but lower catalytic efficiency towards 4MU-?-(GlcNAc)2 than OfCht?-?2. OfCht?-1 had lower affinity towards 4MU-?-(GlcNAc)3 and the ethylene glycol chitin than OfCht?-?2, but their catalytic efficiency was same. The substrate specificity of both OfCht?-1 and OfCht?-?2 towards (GlcNAc)n (n=2-6) was decreased with the decreased degree of polymerization. And both OfCht?-1 and OfCht?-?2 could not hydrolyze (GlcNAc)3 and (GlcNAc)2. The hydrolysis activities of both OfCht?-1 and OfCht?-A2 towards ethylene glycol chitin were much higher than colloidal chitin and a-chitin. Besides, OfCht?-1 had no synergistic effect with OfCht?-2 or OfCht?-?2 towards ethylene glycol chitin, colloidal chitin and a-chitin.(4) The result of Western blot demonstrated that the cleavage of OfCht? occurred in the yeast cell and the reason may be the hydrolysis of proteases during the process of secretory expression. First method was modifying culture by adding protease inhibitors and casamino acid. Second method was constructing the target gene into protease defect P. pastoris strain SMD1168. And the third method was mutanting the Lys and Arg in the linker to Asn. Unfortunately, all the three methods could not obtain the full-length OfCht?.(5) The full-length protein OfCht?-DGY was obtained. The target gene was constructed into an intracellular expression vector pPIC3.5k. The recomibinant OfCht?-DGY was purified by chelating affinity chromatography, however, the purity was low. Using 4MU-?-(GlcNAc)2 as substrate, the target protein was proved in the 150 mM fraction. Using gel filtration chromatography, the protein was further purified and a protein with molecular weight of 92.4 kDa was obtained. Through Western blot, Native PAGE and substrate gel electrophoresis, the protein was proved to be the full-length OfCht?-DGY. However, the expression quantity was very low and the expression condition needed further optimization.