Gluconobacter oxydans NH-10中短链D-阿拉伯糖醇脱氢酶的研究  

Short-chain D-arabitol Dehydrogenase from Gluconobacter oxydans NH-10

作　　者：戴小燕[1] 沈晓波[1] 朱宏阳[1] 徐虹[1] 

机构地区：[1]南京工业大学食品与轻工学院材料化学工程国家重点实验室,南京210009

出　　处：《中国生物工程杂志》2010年第11期39-43,共5页China Biotechnology

基　　金：国家"973"计划(2007CB14304);国家自然科学基金(20906050);江苏省属高校自然科学重大基础研究(08KJA180001);江苏省高校自然科学研究项目(09KJB530007);江苏省博士生自然科学类科研创新计划(CX09B_143Z);江苏省博士后基金(0901012B)资助项目

摘　　要：以氧化葡萄糖酸杆菌(Gluconobacter oxydans)NH-10基因组DNA为模板,扩增得到D-阿拉伯糖醇脱氢酶基因arDH,将其克隆到大肠杆菌表达载体JM109(DE3)中进行诱导表达。SDS-PAGE电泳分析ArDH的分子量约为30kDa,是一个短链脱氢酶,既能催化D-阿拉伯糖醇氧化为D-木酮糖,又能催化D-木酮糖还原为D-阿拉伯糖醇。催化氧化反应时,对D-阿拉伯糖醇的Km为60.67mmol/L,Vmax为0.803U/mg;它能同时依赖于NAD+和NADP+,但是更加偏好辅酶NAD+;最适pH为12.0。还原反应对D-木酮糖的Km为36.39mmol/L,Vmax为1.71U/mg;最优pH为7.0,最适温度均为30℃。Based on combination of bioinformatics,The similarity between the cloning gene and corresponding gene of Acetobacter suboxydans reach to 80%.Then,recombinant plasmid was constructed by inserting arDH genes into vector pET22b and functionally expressed into E.coli JM109（DE3）.The molecular mass of recombinant D-arabitol dehydrogenase was about 30 kDa,so it belonged to the short-chain dehydrogenase.The recombinant enzyme was purified by His Trap and subjected to enzymological characterization.The ArDH could not only oxidize D-arabitol to D-xylulose,but also reduce D-xylulose to D-arabitol.When catalyzing oxidative reaction,the Km value for D-arabitol was 60.67 mmol/L,Vmax was 0.803 U/mg;it could use NAD＋ and NADP＋ as cozyme,but it preferred to depending on NAD＋;the optimum pH and temperature of oxidative reactions was 12.0 and 30℃.However,when catalyzing reductive reactions,the Km value for D-xylulose was 36.93 mmol/L,Vmax was 1.71 U/mg;the optimum pH and temperature of reductive reactions was 7.0 and 30℃.

关 键 词：氧化葡萄糖酸杆菌 D-阿拉伯糖醇脱氢酶 克隆 

分 类 号：Q815[生物学—生物工程]