嗜热芽孢杆菌丙酮酸激酶和乳酸脱氢酶的基因克隆、蛋白表达纯化及活性分析  

Gene Cloning,Protein Expression,Purification and Activity Analysis on Pyruvate Kinase and Lactate Dehydrogenase from Thermophilic bacillus

作　　者：王文珍 王慧慧 陈美雯 徐如飞 蒋培蓉 蒲首丞[1] 巩菊芳[1] 孙梅好[1] WANG Wenzhen;WANG Huihui;CHEN Meiwen;XU Rufei;JIANG Peirong;PU Shoucheng;GONG Jufang;SUN Meihao(College of Chemistry and Life Sciences,Zhejiang Normal University,Jinhua 321004,China)

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004

出　　处：《山西农业科学》2019年第4期580-584,593,共6页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金项目(30800019);浙江省重中之重学科生物学开放基金项目(2017KFJJ001)

摘　　要：偶联法测定酶的活性是常用酶活测定方法之一。核苷二磷酸(NDP)是核苷三磷酸的β,γ高能磷酸键水解产物之一,丙酮酸激酶(PK)和乳酸脱氢酶(LDH)是用来测定NDP生成的常用偶联酶。为了分析嗜热菌苍白空气芽孢杆菌(Aeribacillus pallidus JH-7)(最适生长温度为50℃)的PK和LDH是否可以作为50℃条件下的偶联酶,研究从Aeribacillus pallidus JH-7基因组中克隆pk和ldh,连接到表达载体pET24a。结果表明,经诱导表达、纯化得到纯度达95%以上的Ap PK和Ap LDH。并利用分光光度法,分别对Ap LDH和Ap PK的动力学常数及催化效率进行了分析。结果表明,Ap PK对NDP的最适底物为ADP,催化效率为5×104 mol/(L·s),最适p H值为8.0,最适温度为45℃;NADH为Ap LDH的最适底物,催化效率为3.7×105 mol/(L·s),最适pH值为5.8,最适温度为35℃。虽然50℃不是Ap LDH和Ap PK的最适温度,但它们在此温度下的kcat分别为16.4,64.5 s-1,暗示它们可以作为此温度下的偶联酶。The enzyme coupling to measuring enzyme activity is one of the commonly method for enzyme activity assays. Nucleoside diphosphate (NDP) is one of the high energy phosphate bond hydrolysates of nucleoside triphosphate. Pyruvate kinase (PK) and lactate dehydrogenase(LDH) are commonly coupling enzymes used to assay NDP production. To analyze whether PK and LDH of the thermophilic bacteria Aeribacillus pallidus JH-7(optimal growth temperature was 50 ℃) could be used as a coupling enzyme at 50 ℃, we cloned pk and ldh from Aeribacillus pallidus JH-7 genome, and they were constructed into the expression vector pET24a. By induction and purify, we got ApPK and ApLDH with a purity of over 95%. The paper respectively analysed kinetic constants and catalytic efficiency of ApLDH and ApPK by spectrophotometry. The results showed that the optimum substrate for ApPK was ADP, and the catalytic efficiency was 5×10^4 mol/(L·s), the optimum pH was 8.0, and the optimum temperature was 45 ℃. NADH was the optimum substrate for ApLDH, the catalytic efficiency was 3.7 × 10^5 mol/(L·s), the optimum pH was 5.8, and the optimum temperature was 35 ℃. Although 50 ℃ is not the optimum temperature for ApLDH an ApPK, but at this temperature, Kcat was 16.4, 64.5 s-1, respectively, indicating that they can be used as coupling enzyme at this temperature.

关 键 词：嗜热菌 丙酮酸激酶 乳酸脱氢酶 原核表达 酶活性测定 

分 类 号：Q55[生物学—生物化学]