摘要
胆固醇氧化酶专一性催化胆固醇为胆甾-4-烯-3-酮,广泛的应用于临床以及食品加工行业。本论文将来源于Pimelobacter simplex的胆固醇氧化酶PsCO4,分别转化到大肠杆菌宿主BL21（DE3）、Rosetta（DE3）和C41（DE3）中,在不同温度（15℃、25℃、37℃）及IPTG诱导浓度（0.01mmol/L、0.1mmol/L、0.5mmol/L）下异源表达Ps CO4。结果表明,转入Rosetta（DE3）菌株的PsCO4蛋白,在IPTG浓度为0.1mmol/L、15℃下经18h诱导表达,PsCO4可溶性表达量最高（0.63mg/ml）。异源表达的胆固醇氧化酶PsCO4最适温度为30℃,最适pH为7.5。通过TLC,GC-MS检测出Ps CO4催化胆固醇生成胆甾-4-烯-3-酮。以胆固醇和β-谷甾醇、豆甾醇和孕烯醇酮为底物,测定PsCO4对四种底物的催化反应动力学参数,胆固醇k（cat）/Km为0.08s-1·μM-1分别高于β-谷甾醇（0.04s-1·μM-1）、豆甾醇（0.005s-1·μM-1）和孕烯醇酮（0.02s-1·μM-1）。
Cholesterol oxidase,which catalyzes the reaction of cholesterol to cholest-4-en-3-one,is widely used in clinical and food processing industry.Cholesterol oxidase from Pimelobacter simplex（PsCO4）was transformed into E.coli BL21（DE3）,Rosetta（DE3）,and C41（DE3）.The bacteria were induced at different temperatures（15℃,25℃,37℃）,and different IPTG concentrations（0.01mmol/L,0.1mmol/L,0.5mmol/L）.The results showed that PsCO4 was expressed greatly in supernatant of Rosetta（DE3）（0.63mg/ml）,at the conditions of 0.1mmol/L IPTG and 15℃.The optimum temperature and p H of heterologous expressed PsCO4 was 30℃ and 7.5.The product of cholesterol catalyzed by PsCO4 was identified by TLC and GC-MS.The substrate specificity of PsCO4 towards cholesterol,β-sitosterol,stigmasterol and pregnenolone was determined.The Km/kcat value of cholesterol（0.08s-1·μM-1）is higher thanβ-sitosterol（0.04s^（-1）·μM-1）,stigmasterol（0.005s-1·μM-1）and pregnenolone（0.02s^-1·μM^-1）.
作者
郭倩倩
高登科
程晓涛
路福平
田之仓优
秦慧民
GUO Qian-qian;GAO Deng-ke;CHENG Xiao-tao;LU Fu-ping;Masaru Tanokura;QIN Hui-min(State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology Ministry of＂ Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, Chin)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第6期34-42,共9页
China Biotechnology
基金
国家自然科学基金(31771911)资助项目
关键词
胆固醇氧化酶
催化活性
酶学性质
底物特异性
Cholesterol oxidase
Catalytic activity
Enzymatic property
Substrate specificity
