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Research on Cloning and Expression of the Bile Salt Hydrolase Gene from Lactobacillus Plantarum JPP2

Author: ZhaoShiZuo
Tutor: GuoBenHeng
School: Shanghai Ocean University,
Course: Food Science and Engineering
Keywords: Lactobacillus plantarum Bile salt hydrolase Clone Expression
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 33
Quote: 0
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Abstract


Bile salt hydrolase (Bile salt hydrolase, BSH, EC 3.5.1.24) is a microbial growth, reproduction process produces a metabolite. Bile salt hydrolase is a cholesterol-lowering effect of lactic acid bacteria in the process of a key enzyme, the enzyme can hydrolyze bound taurocholate glycine salts and bile salts, which was converted into amino acids and free bile acids. At present, many BSH has been purified from a variety of microbes and identification, such as Lactobacillus, Bifidobacterium, Enterococcus, Clostridium, Bacteroides and Streptococcus. Domestic and international clinical trials, probiotics and related products have reduced human serum cholesterol, serum cholesterol fall from the sky and lactic acid produced by BSH activity are closely related. This article from Lactobacillus JPP2 section of chromosome encoding bile salt hydrolase gene were cloned and sequenced bsh3 analysis, found to have five conserved bile salt hydrolase amino acid active sites were Cysteine ​​(C) -2, Arginine ( R) -18, Aspartic acid (D) -21, Asparagine (N) -175, Arginine (R) -228. NCBI on with the same five genus Lactobacillus strains were compared and found amino acid sequence homology reached 99%. Using the intermediate cloning vector pMD19-T to construct an expression vector pET-28b and transformed into the expression host E.coli BL21 (DE3), recombinant BSH successful engineering bacteria. As pET-28b vector carries a strong T7 promoter, IPTG induction, high-level expression of exogenous gene easily lead to the generation of inclusion bodies, cells were collected by high-pressure homogenizer lysing by polyacrylamide gel electrophoresis and difference microscope, concluded lysing insoluble material containing inclusions. Ni-chelating column by successfully isolated and purified recombinant bsh3 bile salt hydrolase, purified recombinant BSH single strip, to electrophoretic homogeneity. The recombinant expression vector and extracts induced extracellular crude enzyme solution. Using High Performance Liquid Chromatography its enzyme activity. On salt glycocholate, taurocholate deoxycholate, glycyl goose bile salt deoxycholate combination of three enzymatic reactions, enzyme activities were: 30.75 U / mL, 32.52 U / mL, 28.91 U / mL . As people's living standards gradually improved, atherosclerosis, coronary heart disease, cardiovascular disease, hypertension disease incidence increased year by year, seriously affecting people's health, and serum cholesterol level is too high then the disease is caused by the above-mentioned series of key factors a. In this study, carried out on a clear bile salt hydrolase enzymatic properties, development of cholesterol-lowering fermented dairy products has important theoretical significance and practical value.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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