屎肠球菌132胆盐水解酶基因的克隆表达与酶学特性

Cloning, Heterologous Expression, and Enzymatic Characteristics of the Bile Salt Hydrolase Gene from Enterococcus faecium 132

摘要:

该研究旨在探究降胆固醇潜在菌株屎肠球菌132胆盐水解酶（bile salt hydrolase，BSH）的活性，通过PCR技术克隆目的基因并在大肠杆菌BL21中表达，同时探讨其酶学特性。利用茚三酮法对其菌体破碎液BSH活性进行测定，比酶活达0.55 U/mg。通过克隆其目的基因，连接pET-28a(+)的表达载体并进行异源表达，聚丙烯酰胺凝胶电泳（SDS-PAGE）实验显示，该酶分子量约37 ku。当底物为甘氨胆酸钠（glycocholic acid，GCA）、pH 5.50时，BSH酶活达4.93 U/mg。酶学特性结果表明，GCA为底物、pH 5.50、温度为30 ℃时达最适条件，BSH水解能力最强，酶活达6.59 U/mg，温度在4~30 ℃时，pH为5~7有较好的热稳定和pH稳定性。不同离子对酶活的影响实验表明Na+、Ca2+、Mg2+均对酶活有激活作用；Mn2+、Fe2+对酶活影响不大，但Ni2+、Al3+、Li2+、Co2+、Zn2+均对酶活有强抑制作用，比酶活降到10%以下，几乎丧失活性。通过酶反应动力学得出该酶的Vmax为6.44 μmol/(min•mg)，Km值为3.16 mmol/L。因此，通过bsh基因克隆并异源表达，提高了屎肠球菌132BSH活性，并且BSH水解各类胆盐能力较强，为降胆固醇功能食品的开发与应用奠定基础。

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.