Mining And Verification Of Oligosaccharide Synthesis-Related Genesfrom Four Lactobacillus

With the rapid development of Chinese economics and the improvement of living standards, people are getting more and more concerned about the environment and health, making the problem of food safety becoming the focus of the whole society. The green and natural diet concept has been formed and widely spread. Meanwhile, functional oligosaccharide and L-methionine have raised attention due to their diverse and unique functions. The disorder of intestinal microecology is one of the important factors that causes health problems to people or animals. Even though functional oligosaccharide cannot be absorbed after entering the body, it can also be utilized by intestinal probiotics and multiplies in intestinal tract thus inhibits the growth of pathogenic microorganism and improves micro-ecological environment. L-methionine is one of the essential amino acids for human. The demand for it is very small, but L-methionine and its metabolites are involved in the body's metabolism thus improve immunity and antioxidant capacity, reduce dependence on antibiotics.With the biotechnology entering into the 21 st century, the second generation sequencing technology is being widely used because of its high throughput, high precision and rich amount of information. Functional gene mining tools based on the second-generation whole genome sequencing has became the focus of research. Organism genetic information carried by the whole genome containes enormous potential research and economic value, and mining gene function has become an important mean of use.Therefore, bioinformatics analysis was carried out to dig the functional gene based on the whole genome sequencing information of Lactobacillus screened by our team and NCBI genome database information taking the gene concerning the synthesis of functional oligosaccharides and L-methionine to verify the object.In this study, functional oligosaccharides and L-methionine synthesis related gene from lactic acid bacteria genome sequence information was dug out by bioinformatics methods, ?-glucosyltransferase(?-GTF-pla??-GTF-cas??-GTF-bre), rhamnosyltransferase(RHLase), ?-fructofuranosidase(?-FFase), sucrose phosphorylase(SPase), homoserine-O-succinyl transferase(Met A), the corresponding genes were cloned by PCR method. The E. coli expression system systems were constructed to verify its function, but the soluble protein with biologically active was not obtained, the products were in inclusion body form except ?-FFase and SPase. Since the soluble ?-GTF-pla was not obtained by induction condition optimization and refolding, Lactococcus lactis expression system was constructed for further functional verification and successful expression of ?-GTF-pla. The functional verification indicated that ?-GTF-plaand ?-FFase was not capable of digesting sucrose, while SPase was consistent with its function. This indicates that digging of functional gene by bioinformatics analysis based on the whole genome information still remains some limititation.SPase was used to synthesis the functional oligosaccharides. When screening SP reaction substrate from maltose, xylose, rhamnose, D- galactose, D-galactose, arabinose and mannose, only D-galactose and maltose could form the significant oligosaccharides, which was a little different from the known acceptor of SPase. Melibiose synthesized by D-galactose and saccharose was used for processing optimization and response surface analysis. Under the condition of 60% substrate concentration, 1:2 substrate ratio, the reaction temperature 40?, and addition of 400 ?L(0.39 U/L) purified enzyme, the largest production of melibiose reached 34.20±0.92g/L after 24 hours.