Reseach On The Characteristics And Functions Of Gene WANG₁108 And WANG₀840 From Lb.Kefiranofa Ciens ZW3

The exopolysaccharide ?EPS? production by lactic acid bacteria?LAB? has the broad application prospect. Compared to other polysaccharides, it is more safe, reliable and functional, such as: antitumor, antioxidant, cholesterol-lowering and immunostimulatory,when used as natural thickener and texture modifier in food industry. Due to the low yield exopolysaccharides by LAB. it become the research hotspot all over the world to improve the EPS production.In previous study, a high EPS yielding strain of Lactobacillus kefiranofaciens ZW3 was isolated from Tibet kefir grain in our lab and the highest yield reached 2000 mg / L.Then, an mutant named 2# with the EPS yield reduced 40% compared with the wild strain was obtained. In order to explore the reasons for the EPS yield decline of 2# mutant,comparative genome analysis of wild and mutant strains by bioinformatics methods. In total, there were 60 genes mutated in CDS or the promoter regions, and six of them have a certain enzyme catalytic domains at least. The six genes were WANG₀173?WANG 0174 ? WANG₀175 ? encoding dihydroxyacetone kinase or its subunit?,WANG 1108 ?serine threonine protein kinase?, WANG₀292 ?beta-galactosidase small subunit?, WANG₀840 ?udp-n-acetylmuramoylalanine-d-glutamate ligase, MurD? where the WANG₀292 is synonymous mutation. The results showed that the relative expression of WANG₁108 and WANG₀840 was significantly down-regulated by RT-qPCR. Therefore,these two genes as the related target genes o EPS synthesis for the next step of molecular mechanism research was chosed.WANG₀840 consisted of a-helix, P-sheet and random coil and belonged to non-secretory protein. The gene was expressed in E.coil BL21 ?DE3? with plasmid pET28a as plasmid vector to identify the function of WANG₀840. E. coli BL21 ?pET28a-ZW3 / 2 # -0840? was detected by SDS-PAGE.WANG₁108 belonged to non-secretory protein and the result of enzyme catalytic activity concentariton showed that the mutant STPK activity was 37% lower than that of the wild strain, indicating that the mutation of WANG₁108 was one of the reasons for the decrease of EPS production.