兔肝辅酶Ⅱ依赖性视黄醛还原酶在大肠杆菌中的表达及其对维生素K3的还原作用和产物的鉴定.docVIP

兔肝辅酶Ⅱ依赖性视黄醛还原酶在大肠杆菌中的表达及其对维生素K3的还原作用和产物的鉴定 作者：肖李锋,刘戈飞,宋旭红,谢健平,张巧霞 单位：汕头大学医学院分子生物学中心，广东 汕头 515041 【摘要】 目的：检测兔辅酶Ⅱ依赖性视黄醛还原酶(NDRR)对维生素K3的催化活性并对该酶还原K3所生成的产物进行鉴定。方法：构建含6times;His标签的兔NDRR的表达菌株，诱导表达并超声裂解后，通过Ni2+亲和层析纯化得到重组兔NDRR。在相对去氧隔绝空气反应体系以及存在高浓度十二烷基磺酸钠的反应条件下，利用高效液相色谱(HPLC)对兔NDRR还原K3的催化活性进行酶动力学研究。利用化学合成，HPLC及荧光光谱扫描对其还原产物进行鉴定。结果：诱导表达后的细菌裂解上清液经一步亲和层析后得到单一酶蛋白，其对K3的比活性分别是原空白菌裂解上清液和经诱导表达后的菌裂解上清液的比活性的18.6和7.3倍。经过HPLC测定，兔NDRR对K3的Km值为30.7，Vmax 0.046mu;moL/(minbull;mg)。利用四氢硼酸钠与K3化学合成的产物进行HPLC分析，其保留时间与酶反应所产生的产物峰保留时间一致，而且反应后短时间内能利用荧光光谱扫描得到K4相应的光谱图。结论：兔NDRR对K3存在还原活性，并生成K4。 【关键词】 兔辅酶Ⅱ依赖性视黄醛还原酶;维生素K3;维生素K4;高效液相色谱 　　Rabbit Liver NADP(H)-Dependent Retinal Reductase Expressed in Escherichia coli: Reduction of Menadione and Product Identification 　　XIAO Li-feng, LIU Ge-fei, SONG Xu-hong, XIE Jian-ping, ZHANG Qiao-xia, LI Rui, HUNAG Dong-yang 　　Center for Molecular Biology, Shantou University Medical College, Shantou 515041, China 　　［Abstract］Objective: To detect the reduction of rabbit NADP(H)-dependent retinal reductase(NDRR)on menadione， and to identify its reductive production. Methods: Rabbit-NDRR was expressed in BL21-AITM with 6times;His-Tag. The recombinant protein was purified by affinity chromatography on Ni2+-NTA-agarose after the transformed cells were sonicatied. The kinetic parameters of rabbit-NDRR were determined by HPLC under anaerobic condition and high-concentration of sodium dodecylsulphate(SDS). The reductive production was identified by chemical synthesis, high performance liquid chromatography(HPLC)as well as fluorescent scanning. Results: The homogeneous enzyme was obtained on a single step by affinity chromatography from the supernatant of transformed cells, and its reductive activity of menadione was 18.6-fold of the supernatant of the empty-transformed cells and 7.3-fold of transformed cells. The Km and Vmax detected by HPLC were 30.7 and 0.046 mu;moL/(minbull;mg)respectively. The retention time of the product menadiol produced by menadione and NaBH4 or reducted b