基于温控载体表达胆固醇氧化酶

Expression of Cholesterol Oxidase with Temperature Induced Vector pHsh

摘要:

胆固醇氧化酶在医药检测、食品加工、农业生产等方面都有重要的应用价值。以来源于Rhodococcus sp.的胆固醇氧化酶基因为研究对象，构建于温控表达载体pHsh上，免去了诱导剂的使用，采用温度调控使胆固醇氧化酶基因在大肠杆菌中成功表达。在摇瓶水平对酶的表达量进行优化，最终的酶量为 855 U/L。使用镍柱将酶纯化，得到单一条带，相对分子质量约为55 kDa。分析其酶学性质，最适pH 7.0，在pH 5.0~7.0稳定；最适反应温度40 ℃，在50 ℃保温，其半衰期为6.9 min。以胆固醇为底物，在pH 7.5、37 ℃条件下测酶活，计算得动力学参数Km为3.38 mmol/L，kcat /Km为6.09 s-1·（mmol/L）。

Abstract:

Cholesterol oxidase is of great value to be applied in medicine，food and agriculture. In this study，cholesterol oxidase from Rhodococcus sp. was constructed into the temperature-induced vector pHsh and then by controlling the culture temperature，the gene was successfully expressed in E.coli without adding inducer. The fermentation optimization of cholesterol oxidase was carried out in the shake flask level. With the optimum condition，the enzyme activity reached 855 U/L. When purified to homogeneity using Ni-NTA，the resulting enzyme had the molecular mass of 55 kDa，the optimal pH 7.0 and temperature 40℃. The enzyme was also stable over a pH range of 5.0~7.0，with t1/2 of 6.9 min at 50 ℃. With cholesterol as substrate at pH 7.5 and 37 ℃，the kinetic parameters Km and kcat/Km were 3.38 mM and 6.09 s-1·mM-1，respectively.