Burkholderia cepacia胆固醇酯酶及其分子伴侣基因的串联表达及酶学性质的研究

摘要

In order to achieve the soluble expression of cholesterol esterase,Burkholderia cepacia ST-200 cholesterol esterase was tandem expressed in E.coli by adding SD sequence between the enzyme and its molecular chaperone.Analysis of SDS polyacrylamide gel electrophoresis showed that the soluble expression of cholesterol esterase realized successfully and the total amount of enzyme was 1 077 U/L in shake-flask fermentation.When purified to homogeneity using Ni-NTA,the resulting enzyme had a relative molecular mass of 37 kDa.Secondary structure of cholesterol esterase was analyzed using circular dichroism and the Tm value was measured.The optimum temperature,the optimum pH,thermal stability,pH stability and organic solvent tolerance of cholesterol esterase were studied and it indicated that cholesterol esterase had maximum activity at pH 7.0 and 45 ℃.%为实现胆固醇酯酶的可溶性表达,以Burkholderia cepacia ST-200胆固醇酯酶为研究对象,将酶及其分子伴侣通过人工添加的SD序列在大肠杆菌中进行串联表达.通过SDS-PAGE 电泳分析,胆固醇酯酶成功获得可溶性表达,摇瓶水平的总酶量为1 077 U/L.经过镍柱纯化之后可以获得一条相对分子量约为37 kDa的单一条带.利用圆二色谱解析胆固醇酯酶的二级结构,并测定Tm值,从最适温度、最适pH、热稳定性、pH稳定性及有机溶剂耐受性等方面对胆固醇酯酶进行研究,结果表明该酶在pH为7.0,45 ℃条件下表现出最大活力.