Expression, Purification And Enzymatic Analysis Of Bile Salt Hydrolase From Bifidobacterium In Escherichia Coli

Bile salt hydrolase(EC 3.5.1.24) catalyzes the hydrolysis of conjugated bile salts in vertebrates into deconjugated bile salts and amino acids, which leads to the synthesis of conjugated bile salts from cholesterol in liver cells and reduces serum cholesterol. Due to its prevention of cardiovascular disease, bile salt hydrolase has become one of the research hot spots. However, few researches were done in high efficiency expression of bile salt hydrolase gene and enzymatic properties. Besides, kinetics and mechanism of the enzyme catalytic reaction is still unclear. In this study, Escherichia coli was used to express bile salt hydrolase gene from Bifidobacterium. Then, bile salt hydrolase was purified through Ni-chelating affinity chromatography, and the recombinant enzyme was then characterized and modified to change its substrate specificity. The main results were as results:In order to enhance the production of bile salt hydrolase in E. coli and study the enzymatic properties, E. coli BL21(DE3) was used to express bile salt hydrolase gene from Bifidobacterium infantis KL412. First, bile salt hydrolase gene was amplified by PCR and cloned into expression vector p ET-22b(+). Then, the recombinant plasmid p ET-22b(+)-bsh was transformed to E. coli BL21(DE3). The activities of recombinant bile salt hydrolase of GDCA and TDCA were 135.2 U×m L-1 and 121.3 U×m L-1, respectively, when recombinant bacteria was induced at 20°C with an induction of 1 mmol×L-1 IPTG.Then the recombinant bile salt hydrolase was purified through Ni-chelating affinity chromatography and the expressed product was analyzed by SDS-PAGE, molecular weight of recombinant bile salt hydrolase was 35.0×103. Analysis of enzymatic properties showed that the recombinant bile salt hydrolase has a preference to glycine bile salts, and the activity of recombinant bile salt hydrolase of GCA was 220.1 U×mg-1. Enzymatic kinetics of this purified bile salt hydrolase showed that bile salt hydrolase from Bifidobacterium was allosteric enzyme, and also revealed its differences in positive cooperativity between the enzyme and substrates, catalytic efficiency and substrate preference.After amino acid sequence alignment of Bifidobacterium bile salt hydrolase,saturation mutagenesis was performed on E60 of the recombinant hydrolase by using p ET-22b(+)-bsh as template. Analysis of enzyme activity mutants found that mutants E60 Y, E60 A, E60 F, E60 I, E60 V and E60 D have improved enzyme activity toward conjugated bile salts. Substrate specificity of all mutants were also changed. Compared with wild type bile salt hydrolase, mutant E60 Y exhibited higher enzyme activity of GDCA and TDCA, which were 2.62 and 1.96-fold of the wild strain, respectively. Analyzing the catalytic kinetic parameters of mutants found that the improved catalytic efficiency may be the main reason for the result of higher enzyme activity.