Molecular Cloning, Biochemical Characterization And Inhibitor Discovery Of Group Ⅳ Chitinase From The Insect Ostrinia Furnacalis

Chitin forms exoskeleton, peritrophic membrane (PM) and some other insect structures, so the regulation of its synthesis and degradation is very important for the growth and development of insects. In insect chitin degradation system, chitinase (EC 3.2.1.14) is a key enzyme responsible for hydrolyzing chitin polymer into chitobiose and chitotriose. Insect chitinases are divided into eight groups, of which group IV chitinase is mainly distributed in intestinal tissue with potential roles including PM synthesis and degradation, food digestion and immunity. Because group IV chitinase may act as potential pesticide targets, the research of group IV chitinase on enzymatic properties, structure-function relationships and inhibitors is of great significance.The work in this thesis includes:(1) We obtained cDNA of OfChtIV, the group IV chitinase from insect Ostrinia furnacalis through PCR and RACE. The full length of cDNA is 1380 bp carrying a coding sequence of 1218 bp encoding 405 amino acids. OfChtIV is predicted to be a secreted protein with its N-terminal carrying a signal peptide of 18 amino acids. Phylogenetic analysis demonstrates that OnChtIV from Ostrinia nubilalis is the closest relative to Of/ChtIV.(2) The expression vector of OfChtIV was constructed and introduced into Pichia pastoris GS115 cells. We determined the optimal methanol induction time to be 120 h when the relative specific activity of OfChtIV reached the maximum. Purified recomibinant OfChtIV was obtained through ammonium sulfate precipitation, gel filtration and chelating affinity chromatography.(3) Characterization of enzymatic properties showed that recombinant OfChtIV was N-glycosylated. Optimal pH is both 4.0 using PNP-β-(GlcNAc)2 and colloidal chitin as substrate. Km value is 13.11 mg-mL-1 towards colloidal chitin and 0.21 mM towards pNP-β-(GlcNAc)2, respectively.(4) Both of naphthalimide derivatives and chitosan oligosaccharides inhibit OfChtIV. Inhibitory activity of chitosan oligosaccharides increased as the degree of polymerization increased. IC50 values of (GlcN)5, (GlcN)6 and (GlcN)7 against OfChtIV are 333.5 μM, 60. 1μM and 12.4 μM, respectively.(5) β-N-acetylhexoaminidase is another key enzyme in insect chitin degradation system. Based on the classical inhibitor NGT, we designed a new inhibitor NMAGT against OfHexl, the β-N-acetylhexoaminidase from Ostrinia furnacalis through crystal structure analysis and molecular docking. Ki value of NMAGT against OfHexl is 0.13 μM, which is 600 times lower than that ofNGT.