Study On Fermentation And Purification Of Recombinant Stichopus Japonicus Lysozyme

Lysozyme is widely distributed among eukaryotes and prokaryotes. It catalyzes the hydrolysis of bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogen. The commercial lysozyme, mainly obtained from hen egg white, only functions to gram-positive bacteria, so its application was limited. In recent years, researches of lysozymes from many sources have been done widely, but the report of lysozymes in marine life was very little.E.coli has following characters:a thorough understanding of genetic traits, fast growth, cultivating economy, high level expression, more plasmids and hosts to be selected etc. Through the foreign proteins are often highly expressed in E.coli, at the same time, it is easy to form the unsoluble inclusion body or be degraded by host proteases. At present the renaturation of protein in vitro is widely studied, but the process is often time-consuming, tedious and not economy. So exploring the foreign proteins in the expression of E.coli have high value of academic research and broad application prospect. The main research methods have the choice of the hosts, selected carriers, cultivation conditions and the choice of induction methods.This project mainly focused on fermentation of the genetically engineered bacteria to express the Stichopus japonicus lysozyme (SjLys), i.e. E. coli BL21 (DE3) pLysS/pET 32a(+) SjLys, as well as purification and characterization of its product. The recombinant SjLys was first expressed successfully in soluble form via optimization of fermentation conditions. The results show that, the soluble recombinant SjLys was successful obtaining when the medium was LB/Amp,2% inoculation,20% fill volume, inducing temperature was 25℃, inducing concentration of IPTG was 1.0 mmol/L, sorbitol concentration was 0.4mol/L, and betaine concentration was 2.5mmol/L. The expressed protein was purified by affinity chromatography, gel chromatography and ultrafiltration. It was gained the electrophoretically pure of 95% of the recombinant SjLys protein. The optimal pH and temperature acting on the substrate Micrococcus lysodeikticus was 6.8 and 45 ℃, respectively. The antibacterial activity of the purified recombinant SjLys was analyed. It showed a broad antimicrobial spectrum against the tested strains including Gram-positive and Gram-negative bacteria. The study of SjLys will provide a basis for production of the sea cucumber lysozyme in a large scale.