Heterologous Expression And Characterization Of The Chitin Degradation Enzymes From Locusta Migratoria

Chitin,a polysaccharide consisting of N-acetyl-β-glucosamine residues linked by(3(1,4)glycosidic bond,mainly exist in the insect exoskeleton,peritrophic membrane,intestinal tract,trachea,and the cell walls of fungi,is the second largest biological polymers in nature after cellulose.Chitin degradation enzymes from Locusta migratoria consist of chitinase and theβ-N-acetylglucosaminidase.Chitinase(Cht)is a kind of endoenzyme which belong to glycoside hydrolase 18 family,however,P-N-acetylglucosaminidase(NAG)is a kind of exoenzyme which belong to glycoside hydrolase 20 family.The two kinds of enzymes work together to degrade the chitin to N-acetyl-glucosamine.Chitin degradation enzymes widely present in the molting fluid,midgut of insects,and venom gland of some insects,which play an important role of insect chitin metabolism in the process of growth and development.Chitinase andβ-N-acetylglucosaminidase have been widely recognized as a potential insecticide targets,the degradation of chitin biological waste,and production of N-acetyl-glucosamine.In this study,we heterologously expressed the group I chitinase(LmCht5-1 and LmCht5-2)andβ-N-acetylglucosaminidase(LmNAG1)of Locusta migratoria in SF9 cells using Bac-to-Bac baculovirus expression system,and then characterized kinetic and carbohydrate-binding properties of purified enzymes.The main research content is divided into three parts as follows:1.Heterologous expression and purification of LmCht5-1 and LmCht5-2The open reading frame corresponding to LmCht5-1,LmCht5-2 and LmNAGl were amplified from locust.A pair of primers containing appropriate restriction sites were designed to facilitate directly subcloning of each fragment for the coding sequence into the pFastBacTM-Dual clone vector behind the polyhedrin promoter,and the reverse primers contained "6×His tag" coding sequences before the termination codon to facilitate the targeted protein separation and purification.The recombinant enzymes,LmCht5-1,LmCht5-2 and LmNAQ containing a 6×His tag at the C-terminal were purified by Ni-NTA agarose chromatography,and detected by SDS-PAGE and western blot.2.Characterization of the chitin degradation enzymes from the Locusta migratoria.We performed characterization analysis of purified enzymes.LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums.LmCht5-1 has lower Km value for the oligomeric substrate(4MU-(G1cNAc)3),and higher Km value for the longer substrate(CM-Chitin-RBV)compared with LmCht5-2.LmCht5-1 has higher Kcat/Km value for the oligomeric substrate(4MU-(G1cNAc)3),and lower Vmax/Km value for the longer substrate(CM-Chitin-RBV)compared with LmCht5-2.LmCht5-1 had relatively higher activity against the oligomeric substrate,4MU-(GlcNAc)3,whereas LmCht5-2 exhibited higher activity towards the longer substrate,CM-Chitin-RBV.A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins.LmNAG1 represent low Km value(0.28±0.02 mM),and high kcat/Km value(3215.12±61.98mM-1S-1)for the substrate(4MU-GlcNAc),and indicate LmNAGl can catalyze 4MU-GlcNAc efficiently.Our results suggested both LmCht5-1 and LmCht5-2,which have the critical glutamate residue in region II of catalytic domain,exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates.LmCht5-1 had relatively higher activity against the oligomeric substrate,4MU-(G1cNAc)3,whereas LmCht5-2 exhibited higher activity toward the longer substrate,CM-Chitin-RBV.And LmNAGl exhibited higher activity against 4MU-GlcNAc substrate.These findings are helpful for further research to clarify their different roles in insect growth and development.3.Chitin-binding properties of LmCht5-1 and LmCht5-2 from the Locusta migratoria.Both LmCht5-1 and LmCht5-2 have a signal peptide,a catalytic domain and a linker region.However,LmCht5-1 has a chitin-binding domain(CBD),LmCht5-2 does not have a CBD.In order to determine the role of CBD in LmCht5-1,the chitin binding property of LmCht5-1 and LmCht5-2 was compared by colloidal chitin binding experiment.Bull Serum Albumin(BSA)was used as control.As a result,LmCht5-1 has a chitin-binding domain(CBD)tightly bound to colloidal chitin,but LmCht5-2 does not have a CBD for binding to colloidal chitin.This study successfully expressed the LmCht5s and LmNAG1 by the Bac-to-Bac baculovirus-insect cell expression system and purified by Ni-NTA,characterized kinetic and carbohydrate-binding properties of purified enzymes.It provides theory basis for pest control using chitin degradation enzymes.