Cloning, Heterologous Expression, and Enzymatic Characteristics of the Bile Salt Hydrolase Gene from Enterococcus faecium 132
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Abstract:
The bile salt hydrolase (BSH) activity of a potentially cholesterol-lowering strain, Enterococcus faecium 132, was studied. The target gene was cloned and expressed in Escherichia coli BL21, and its enzymatic characteristics were examined. The BSH activity of E. faecium 132 lysate was determined using the ninhydrin method, and its specific enzyme activity was 0.55 U/mg. The bsh gene of E. faecium 132 was cloned into the expression vector pET-28a(+) for heterologous expression in BL21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of the resulting enzyme was approximately 37 ku. The recombinant BSH showed optimal hydrolytic activity (6.59 U/mg) when glycocholic acid (GCA) was used as a substrate at a pH of 5.50 and temperature of 30 ℃, while also exhibiting good thermal and pH stability within a temperature range of 4~30 ℃ and pH range of 5~7. The effect of different metal ions on enzyme activity indicated that BSH activity was activated by Na+, Ca2+, and Mg2+, unaffected by Mn2+ or Fe2+, but inhibited by Ni2+, Al3+, Li2+, CO2+, and Zn2+, with enzyme activity decreasing to < 10%, and an almost complete loss of enzyme activity. Enzymatic kinetics revealed that the Vmax of this enzyme was 6.44 μmol/(min•mg), and the Km value was 3.16 mmol/L. Therefore, through the cloning and heterologous expression of the bsh gene in E. coli BL21, significant improvements can be made to E. faecium 132 BSH activity, and various bile salts were strongly hydrolyzed, thus implying that this recombinant BSH can be used in the development and application of cholesterol-lowering functional foods.
