Fermentation Of Recombinant E.coli Expressing Lysozyme Genes From Marine Organisms And Study On Expressed Products

Lysozyme is a ubiquitous enzyme that is widely distributed in the organism kingdom. The biological function of this enzyme is believed to be self-defense from bacterial infection because it induces bacterial cell lysis by hydrolyzing β-1,4linked glycosidic bond of the peptidoglycan on the bacterial cell wall. Lysozyme has a good inhibitory effect on Gram-positive bacteria and some Gram-negative bacteria, but has no effect on the cells of human and animal. So it is identified as a non-toxic, harmless and safe additives by WHO and many countries. Therefore, it is more and more widely used in medicine, food, biotechnology and the process of feeding animals.Three families of lysozymes have been identified in animals:c (chicken) type,g (goose) type,and i (invertebrate) type. Recently, several i-type lysozymes have been identified by sequence alignments, and most of these belong to marine bivalve mollusks. The ⅰ-type lysozymes are a special kind of lysozyme and have the double function characteristics. It should be noted that the bivalve lysozymes show striking protein similarity to destabilase, which hydrolyzes the ε-(γ-Glu-)-Lys crosslinkage between Glu and Lys in stabilized fibrin. At the same time, i-type lysozymes also have enzymatic (glycosidase) antibacterial action. Lysozymes from Stichopus japonicus and Crassostrea gigas belong to the ⅰ-type lysozyme. At present, the study of marine organisms mainly concentrated on the breeding, deep processing of products and medicinal activity, whereas the molecular investigation of marine organisms is less reported.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. But 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 soluble proteins heterologously expressed in 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. The aim of the study is to construct two genetically engineering strains for efficient expression of lysozymes from S. japonicus and C. gigas, designated as SjLys and CgLys, respectively. Firstly, the total RNA was extracted from S. japonicus and C. gigas. The gene fragments of SjLys and CgLys were synthesized and amplified from the total RNA by RT-PCR. The resulting products were cloned into the pMD-18T vector and sequenced. The gene length of SjLys and CgLys were378bp and354bp, encoding125and117amino acids, respectively. Secondly, the gene fragments of SjLys and CgLys were cloned into the plasmid pET-32a(+) and transformed into Rosetta(DE3)pLysS to form the recombinant strains, i.e. pET-32a-SjLys/E.coli Rosetta(DE3)pLysS and pET-32a-CgLys/E. coli Rosetta(DE3)pLysS. Thirdly, the two strains was induced to express the target proteins by0.5mM IPTG for4h in25℃as the optimal conditions. As a result, the recombinant SjLys and CgLys proteins were over-expressed but as an insoluble form (inclusion body). Each expressed protein accounted for over80%of the total protein. In order to obtain the active form of the target proteins, many efforts were done to denature, purify and refold the recombinant SjLys and CgLys proteins, howerer, little is achieved.Therefore, another expression vector (pCold-sumo) was used to enhance the solubility of the target proteins. The genes of SjLys and CgLys were connected to the vector pCold-SUMO. The recombinant plasmids were transformed into E. coli Rosetta(DE3)pLysS. After induction by IPTG for24h in15℃, the recombinant proteins of SjLys and CgLys were obtained as a soluble form, but the amount of each expressed protein was about10%of the total protein.The antibacterial activities of the purified recombinant SjLys and CgLys was analyzed and shown a broad antimicrobial spectrum against the tested strains including Gram-positive and Gram-negative bacteria.