The Functional Analysis Of Active Residues In ?-4 Conserved Motif Of Lymantria Dispar Chitinase

The gypsy moth is one of the most destructive polyphagous pests in the world with more than 500 host plants.In order to control the harm of gypsy moth,people have tried many methods.In recent years,more and more attentions have been paid to biological control.Chitin is a vital component of the insect cuticle and peritrophic membrane and is also a barrier for insects to prevent biological damage and mechanical damage.During insect growth and development,the cuticle and PM must be degraded periodically and then be replaced,thus the biosynthesis and degradation of chitin played an important role in the growth and development of insects.Insect chitinase is a key enzyme involved in the degradation of chitin,in which Group I chitinase is considered to be a potential green pesticide design target because of its irreplaceable role in pupae to adult metamorphosis.The amino acid residues in the second conserved catalytic region,which has the consensus sequence(F/L)DG(L/I)DLD(W/I)EYP,have been implicated for catalysis process.In order to study the roles of D143,D145 and W146 and their relationships,site-directed mutagenesis on D143,D145 and W146 in the conserved motif of LdCht5 was studied,and obtained seven mutants in total(D143E,D145 E,W146G,D143E/D145 E,D143E/W146 G,D145E/W146 G,D143E/D145E/W146G).The mutated and wild genes were cloned into the pFastBacTM vector,geting the recombinant pFastBac Dual-LdCht5 and the seven mutant plasmids were transformed into competent E.coil strains DH10 B,generating transfer vector.The verified recombinant transfer vectors were transfected into Sf9 cells using a cellfectin reagent.The protein expression was carried out in suspended Sf9 cells causing the high yields of these enzymes.The enzymatic and kinetic properties of the enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)3.The major findings are as follows:First,among the seven mutants,D145 E,D143E/D145 E,D145E/W146 G mutations still kept some extent catalytic activity toward MU-(GlcNAc)3,while D143 E,W146G,D143E/W146 G,D143E/D145E/W146 G mutant enzymes were inactived.Second,by comparing the enzymatic properties of wild-type and mutant enzymes,the mutants exhibited maximum activity and stability at pH 6 and showed the highest activity at 50 oC which was similar to thewild-type enzyme.But the wild-type chitinase exhibited the narrowest pH range of activity and stability which indicated the mutant enzymes may had better application scope and economic value.Third,compared with all the mutant enzymes,the wild-type enzyme had the highest kcat/Km which means the decreased catalytic efficiency of LdCht5 after the mutation.However,the decreased Km values of mutant enzymes indicated they had a higher affinity toward 4MU-(GlcNAc)3than wild-type enzyme.The study of site-directed mutagenesis in Lymantria dispar chitinase will better understanding the role of the critical residues and their interactions,providing theoretical support for the potential pesticid targets.Moreover,chitin hydrolyzate N-acetylglucosamine is widely used in medical,food and chemical applications.In the enzymatic hydrolysis of chitin,the mutant enzymes may have better economic value compared with the wild enzyme.