Expression, Purification And Characterization Of A Chitinase From Lithocolletis Ringoniella

Chitin, a polysaccharide consisting of β(1,4)-linkedN-acetyl-β-glucosamine residues, is an insoluble structural component found in the exoskeleton and gut linings of insects. Insect chitinase, one of18family glycoside hydrolases, is widely present in the midgut of insects, molting gland and some insect venom gland. During insect growth and development, chitinase plays an important roles. So far insect chitinase is mainly used in the degradation of chitin biological waste, disease control and insect pest control.In order to express the LrCHT5in E.coli as a soluble fusion protein, the gene of LrCHT5was cloned into different expression vectors. Different recombinant plasmids were transformed into Corresponding BL21chemically competent cells and induced by IPTG. SDS-PAGE and western blot analysis showed that combine pMAL-LrCHT5could expessed soluble fusion protein, MBP-LrCHT5. Subsequently, the expression conditions of the fusion protein MBP-LrCHT5was futher Optimized. The optimal OD was0.778and the optimal concentraction of IPTG was0.5mM. The Amylose resin was used to purify fusion protein.In order to express the LrCHT5with biological activity, LrCHT5gene was recombined into vector pFastBacTMDual and then transformed into DH10Bac to Bac form Bacmid-LrCHT5which was transfected into Sf9cells afterward. Bacmid-LrCHT5viruses were retrieved from the supernatant and Sf9cells were infected with Bacmid-LrCHT5viruses to express fusion protein LrCHT5-(His)6. SDS-PAGE analysis proved that fusion protein was expressed by Sf9cells and Ni-NTA was used to purify the protein.In this study, the activity of different sources of LrCHT5were detected by a short oligomeric substrate, MU-(GlcNAc)3. The study proved that LrCHT5expressed by prokaryotic expression system had no biological activity while Sf9cell expressed protein had a different result. The optimum pH value of the LrCHT5from Bac-to-Bac Baculovirus Expression System was6.0, and the optimum temperture was50℃. Metal ions like Mn2+, Ca2+, Mg2+and Ba2+played catalytic role while Cu2+, Fe3+and SDS strongly inhibited activities of LrCHT5. The Km and Vmax was14.2μm/L and0.905μM/min, respectively. In addition, LrCHT5significantly inhibited the spore germination of two fungal species, Penicillium and Saccharomyces cerevisiae.