Research On Cloning And Expression Of The Bile Salt Hydrolase Gene From Lactobacillus Plantarum JPP2

Bile salt hydrolase(BSH,EC 3.5.1.24)is a metabolite of growth and reproduce of microbe. BSH is an important enzyme in cholesterol-removing effect of Lactic acid bacteria(LAB) which could hydrolyze conjugated bile salt to amino acid and deconjugated bile salt.From now on many BSHs have been purified and characterized from various microorganisms. Such as Lactobacillus, Bifidobacterium, Enterocococcus, Clostridium and Streptococcus. Both home and abroad clinical trials proved that probiotic can remove serum cholesterol, and its effects are related to BSHs from LAB.The research is mainly about the sequence analysis in bsh3 gene from Lactobacillus plantarum JPP2. The amino acid sequence represents five conserved active site: Cysteine-2, Arginine-18, Aspartic acid-21, Asparagine-175, Arginine-228. Looked for genes that encode BSH enzyme in the Lactobacillus plantarum in public databases from NCBI showed that bsh3 homologs are 99% identity or more. The bile salt hydrolase gene bsh3 was amplified from Lactobacillus plantarum JPP2 by PCR technique. The purified PCR products was inserted into expression vector pET-28b by means of intermedia cloning vector pMD19-T. The positive plasmid was transformed into E.coli BL21(DE3), and recombinant strains E.coli pET28-bsh3 was obtained. T7 enhanced promoter was carried by pET-28b vector, after IPTG induction, high expression of exogenous gene may easily lead to inclusion bodies. Cell was disrupted by high-pressure homogenization,inclusion bodies was founded in insoluble pellet fraction and identify by SDS-PAGE and observed by phase microscopy. The recombined BSH of E.Coli BL21-pET28b-bsh3 in inclusion bodies was purified by Ni2+ affinity chromatography. The purified BSH was single band in SDS-PAGE.The BSH was induced by IPTG, and extract extracellular enzyme. HPLC-based BSH activity assays using glycocholate, taurodeoxycholate and glycochenodeoxycholate as substrates, revealed that bsh3 activity were 30.75 U/mL, 32.52 U/mL, 28.91 U/mL respectively.With the improving gradually of people's living standard, the morbidity arteriosclerosis,coronary heart disease,hypertension are increasing year by year. And high serum cholesterol level is one of the main factors that cause the series of diseases above. The results of this work could provid rationale for functional dairy products and cholesterol-reducing drugs.