Site-directed Mutagenesis Of Amino Acid Residues D143, D145,W146 And E147 In Active Catalytic Domain Of Lymantria Dispar Chitinase(Glass Ⅰ)

Chitin, a polysaccharide long-chain polymer comprised of P (1,4)-N-acetylglucosamine residues, is one of the most important biopolymers in nature. It is mainly exited in fungi, arthropods and nematodes. Insect chitinases belong to family 18 of the glycohydrolase superfamily and are involved in shedding of the old cuticle and turnover of the peritrophic membrane (PM) of insects during molting. During the normal growth of insects were effected once abnormal expression pattern of chitinases in different tissue or stage of development. Studying the functions of key amino acids of insect cintinase plays an important role in the study of catalytic mechanism of enzymes.In this study, we first cloned chitinase gene (Glass I) from Lymantria dispar(LdCht5), the gene (LdNSD) with no signal peptide coding region and the gene with active site coding region (LdCD) into different prokaryotic expression vectors and eukaryotic expression vectors respectively for the selection for the best expression vectors, with expectation that the resulting target protein expressed in these vectors would have biological activity and proper molecular weight for its subsequent purification, which would lay a solid foundation for its later catalytic site study. We used pET28a, pET32a and pGEX-4T-1 as prokaryotic expression vectors and pFastBacTMDual as eukaryotic expression vector. The recombinant prokaryotic expression vectors (pET28a-LdCht5, pET28a-LdNSD, pET28a-LdCD, pET32a-LdCht5 pET32a-LdNSD, pET32a-LdCD, pGEX-LdCht5, pGEX-LdNSD, pGEX LdCD) were expressed in E. coli expression system with IPTG as inducer, and the recombinant bacmids (Bacmid-LdCht5, Bacmid LdNSD, Bacmid LdCD) were expressed in the baculovirus-infected insect cell system. The results from SDS-PAGE electrophoresis analysis showed that the proteins expressed in E. coli system were present in the form of inclusion body, and even with induced expression at low temperature overnight, the expressed proteins were still present in the form of inclusion body, whereas the expression products from the three recombinant bacmids in the insect cell system were present in soluble form. Using 4-MU-(GlcNAc)3 as the substrate to measure the activity, the expression product from Bacmid-LdCht5 had biological activity, while the expression products from Bacmid-LdNSD and Bacmid-LdCD had no any biological activity, therefore the subsequent mutation study of the catalytic site would be based on LdCht5.The insect cell expression system was chosen as expression system for this site-directed mutation study of the catalytic site. The four amino acids of FDGLDLDWEYP on the catalytic site of chitinase gene (Glass I) from Lymantria dispar(LdCht5) were mutated to give four mutant recombinant bacmids(Bacmid-W146G, Bacmid-D143E/W146, Bacmid-D143E/D145E/ W146G, Bacmid-D143E/D145E/W146G/E147D), which then were transfected by using a transfection reagent into sf9 cells for expression. Since LdCht5 had a signal peptide, the nascent peptides could be correctly secreted into the extracellular medium. The medium was collected and dialyzed, and the protein was separated and purified with Ni-NTA column, The single band from the SDS-PAGE electrophoresis indicated that that the protein obtained was relatively pure, which could facilitate the subsequent study on the enzymatic properties of each mutant protein.In the activity study of the mutant proteins, the activity of decomposing the subtract of chitinase gene (Glass I) mutant, W146G, at pH 3 to 10 and temperature 30 to 60℃ was not detectable, which indicated that if the change of W146 into W146G occurred in conservative sequence, chitinase gene (Glass I) from Lymantria dispar(LdCht5) would lose its activity. When W146G was further mutated into D143E/W146G, its activity was still very low or even not detectable. But when the mutant D143E/W146G was mutated into mutant D143E/D145E/W146G, its activity increased greatly. In comparison with its wild type, its enzymatic activity was 70% of the wild type, which probably suggested that the simultaneous change of several amino acids might have a concert effect. When the D143E/D145E/W146G was still further mutated into the mutant D143E/D145E/W146G/E147D, its activity lost again.In this study, we investigated the catalytic site of chitinase gene (Glass I) from Lymantria dispar(LdCht5), an agricultural pest, using molecular biology methods and downstream processing techniques in biological engineering. By testing the eukaryotic and prokaryotic expression systems, we finally chose the baculovirus-insect cell expression system as our expression system. To the best of our knowledge this is the first study on the catalytic site of chitinase gene (Glass I) from Lymantria dispar(LdChl5). The results from this study will enhance an understanding of chitinase gene (Glass I) and its catalytic site, and will provide a theoretical basis for developing methods for the biological control of Lymantria dispar.