嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究  

Cloning,expression and characterization of NAD kinase from Geobacillus stearothermophilus

作　　者：侯堃[1] 李红梅[1] 侯立琪 

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093 [2]上海立瞻生物科技有限公司,上海200000

出　　处：《工业微生物》2014年第6期39-44,共6页Industrial Microbiology

基　　金：上海市研究生创新基金(编号:54-13-115-103)

摘　　要：NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a(+)为表达载体、E coli BL21(DE3)为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816 bp,酶分子量大约为35 kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH 7.5,在35℃中保温2 h后仍能保持80%左右的活性。Mn^(2+)、Ca^(2+)对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4.43 U/mg。动力学性质分析结果显示NAD激酶对底物NAD催化的K_m和K_(max)分别为1.46 mmol/L和0.25μmol/(L·min)。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。NAD kinase can catalyze NAD to form NADP. The NADK gene coded NAD kinase from Geobacillus stearothermophilus was obtained by PCR in this study. Then the NADK gene was overexpressed in E. coli BI21 （ DE3 ） with plasmid pET30a （ ＋ ）. The results showed that an 816 bp NADK gene was attained from Geobacillus stearothermophilus, and the molecular weight of NAD kinase was about 35 kD. Furthermore, the enzymatic characteristic of NAD kinase indicated that the optimal temperature and pH were 35℃ and pH 7.5, separately. And 80% enzyme activity could maintain even after 2 h of heat treatment at 35℃. Enzyme activity could be activated by Mn2＋ and Ca2＋ dramatically. Under the above optimum conditions, the enzyme activity of NAD kinase could reach 4.43 U/mg. When NAD was used as substrate, the enzyme kinetic constants of Kmand Vmax was 1.46 mmol/L and 0.25μmol/ （ L · min）, respectively. The NAD kinase from Geobacillus stearothermophilus expressed in E. coli BL21 （DE3） could provide more biological resources for the biosynthesis of NADP.

关 键 词：嗜热脂肪地芽孢杆菌 NAD激酶 克隆表达 酶学性质 

分 类 号：Q936[生物学—微生物学]