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

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℃.