阴沟肠杆菌铁氢酶(hydA)基因在大肠杆菌中的表达及其产氢特性  

EXPRESSION OF Enterobacter cloacae hydA GENE IN E.coli AND THE FERMENTATIVE HYDROGEN PRODUCTION OF THE RECOMBINANT E.coli

作　　者：赵锦芳[1] 程军[2] 戚峰[2] 张传溪[1] 

机构地区：[1]浙江大学昆虫科学研究所分子生物学实验室,杭州310029 [2]浙江大学能源清洁利用国家重点实验室,杭州310027

出　　处：《太阳能学报》2009年第6期829-832,共4页Acta Energiae Solaris Sinica

基　　金：国家高技术研究发展计划(863)项目(2006AA05Z122);国家自然科学基金(50406022);全国优秀博士学位论文作者专项资金(200437)

摘　　要：合成Enterobacter cloacae IIT-BT 08的铁氢酶(hydA)基因片段,PCR扩增出全长ORF。将目的基因克隆到原核表达载体pGEX-4T-2,构建了表达质粒pGEX-4T-2-hydA,转化大肠杆菌BL21(DE3)并在IPTG诱导下表达。SDSPAGE分析表明在约42kDa处有特异性条带。用抗GST多克隆抗体对GST-hydA融合蛋白进行了Western blot检测,进一步证明了hydA得到了正确的融合表达。产氢活性检测结果表明表达的重组hydA在大肠杆菌中具有明显的产氢活性。测定了培养基初始pH值、温度和底物浓度对重组大肠杆菌产氢的影响。The coding region of hydA gene from Enterobacter cloacae IIT-BT 08 was amplified by using PCR method from a synthesized template. The cloned coding region of hydA was inserted into the expression vector pGEX-4T-2 to form the recombinant plasmid pCEX-4T-2-hydA and was then transformed into E. eoli BL21 for expression. SDS-PAGE analysis showed that an expression band about 42kDa and this GST-hydA fusion protein was confirmed by western blot analysis us- ing the polyelonal antibody against GST. The hydrogen production tests show that the recombinant hydA in E. eoli has the bioaetivity. Effects of the initial pH value, temperature and glucose concentration on the fermentative hydrogen pro- duction using the recombinant E. coli were further characterized.

关 键 词：氢气 阴沟肠杆菌 铁氢酶 原核表达 发酵 

分 类 号：Q7[生物学—分子生物学] TQ116.29[化学工程—无机化工]