Cloning, Expression And Characterization Of A Bile Salt Hydrolase From Lactobacillus Fermentum DF-4

Gilliland issue that lactobacillus can reduce the content of cholesterol in1977. And then, abundance of research confirm and delves into this, and proposed generally that the Bile Salt Hydrolase(BSH) play an important role in the content reduction. BSH is a hydrolase which can hydrolyze conjugated bile salt into unconj ugated bile salt and ami no acid, and the content of conj ugated bi I e sal t become I ower i n vi vo. Whi ch boost the chol esterol to combi ne and repi eni sh the conjugated bile salt. As a result, the content of cholesterol become lower in body. In this research, we found one lactobacillus which can reduce the content of choledterol efficiently, verify it’s Lactobacillu. Fermentum by16sRNA, and named it L. Fermentum DF-4. There are little of report about the L. Fermentum to reduce cholesterol’content, so the research about DF-4is meritoriousThe gene of BSH EU477374was referenced, program Primer5was used to design the specificity primer of BSH, and the gene fragment was cloned by PCR. The gene fragment from FCR was verified as gene bsh of L. Fermentum with975bp.Gene recombination technology was applied to recombinate gene bsh into expression vectors, and electrotransformated into E.coli. Indudble expressed therecombinant E.coliswith IPTG, and found that the recombinant E.coli Rosetta[pET-28a-BSH] expressed the protein BSH in the form of inclusion body, with weak solubility. But the weak solubility of protein BSH can improve with fusion protein.So fusion protein IF2,、SUMO, GST, NusA and MBP was used, and found that IF2and MBP is suitable with BSH. Analysed with SDS-PAGE, fusion protein MBPis the best one.The ultrasonication was used to break cells and extract primary enzyme liquid. Through probing the best ultrasoni cation parameter, the content of bacteria suspension with0.01g/mL, broken volume with9ml_, working rate5s-10s-5s,200w, and work120times, which is th ebest.In this research, the Ni-NTA Resin was used to purificiation, and the result showed the Ni-NTA Resin bonding with the His-tag at C-terminal of ami no acids can gain more MBP-BSH protein with less other proteins Enzyme was measured by triketohydrindene hydrate, and the enzyme activity was62.365U/mg.Those result can prove the heterologous expression protein of MBP-BSH did have the function of BSH. The report about the BSH combined with fusion protein and it’s enzyme activity was never published before, so it’s the first time.