Truncation And Site-directed Mutagenesis On Catalytic Domain Of Lymantria Dispar Chitinase (Class â… )

Lymantria dispar is a worldwide pest which is distributed around the world and the insect can cause significant impact on forestry. There are some methods to control it, for example, sex pheromone, manual controlling, chemical pesticide spraying and so on. Recent years, the biological control of Lymantria dispar is more and more popular. The peritrophic membrane and cuticle of insects contain chitin, which is a linear polysaccharide of N-acetylglucosamine (GlcNAc) connected with β-1,4 glucosidic bonds. The cyclical generation and degradation of the chitin plays a key role in the insect growth and development process. Chitinase can degrade chitin and it has already been used in the biological control for many years. In this study, the truncated and site-directed mutagenesis on catalytic domain of the Lymantria dispar chitinase were researched. As a result of the research, we can understand the mechanism of chitinase more deeply and provide a theoretical basis for the further using of the insecticide.In this study we cloned four truncated domains and site-directed mutagenesis catalytic domains of Lymantria dispar chitinase (LdCHTS) by PCR. Four kinds of truncated genes were cloned, including LdCHT5-N20, LdCHT5-N60, LdCHT5-C20, LdCHT5-C60. At the same time, there were six kinds of site-directed mutations, LdCHT5-D143A, LdCHT5-D143E, LdCHT5-D143N, LdCHT5-D145A, LdCHT5-D145E, LdCHT5-D145N. These genes were connected to an eukaryotic expression vector (Bacmid-LdCHT5-N20, Bacmid-LdCHT5-N60, Bacmid-LdCHT5-C20, Bacmid-LdCHT5-C60, Bacmid-LdCHT5-D143A, Bacmid-LdCHT5-D143E, Bacmid-LdCHT5-D143N, Bacmid-LdCHT5-D145A, Bacmid-LdCHT5-D145E, Bacmid-LdCHT5-D145N) and the proteins were expressed using Bac to Bac expression system. In this process, the vectors binding with objective genes were transfected into Spodoptera frugiperda (Sf9) cells by using the cellfectin reagent. These two types of proteins were both purified by nickel column and the purified proteins would provide a basis for further study of enzymatic activity.CM-chitin-RBV was used as substrate to study the enzymatic activity of these proteins. The results showed the four truncated catalytic proteins had no activity and were not conducive to subsequent experiments. The research results of the catalytic domain site-directed mutagenesis areas were:D143N had the highest activity in the six mutants, followed by D143E. The two mutants retained 84%,65% activity of the wild type, respectively. D145E, D145N lost more than half of the activity and D143A, D145A lost almost 90% of the activity.