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

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30 kDa,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K_m为60.67 mmol/L,V_max为0.803 U/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 K_m为36.39 mmol/L,V_max为1.71 U/mg;最优pH为7.0,最适温度均为30℃。

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

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 no...