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

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中国生物工程杂志 ›› 2010, Vol. 30 ›› Issue (11) : 39-43.

研究报告

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

戴小燕, 沈晓波, 朱宏阳, 徐虹

作者信息 +

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

DAI Xiao-yan, SHEN Xiao-bo, ZHU Hong-yang, XU Hong

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文章历史 +

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℃。

Abstract

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 K_m value for D-arabitol was 60.67 mmol/L, V_max 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 K_m value for D-xylulose was 36.93 mmol/L, V_max was 1.71 U/mg; the optimum pH and temperature of reductive reactions was 7.0 and 30℃.

导出引用

戴小燕, 沈晓波, 朱宏阳, 徐虹. Gluconobacter oxydans NH-10中短链D-阿拉伯糖醇脱氢酶的研究[J]. 中国生物工程杂志, 2010, 30(11): 39-43

DAI Xiao-yan, SHEN Xiao-bo, ZHU Hong-yang, XU Hong. Short-chain D-arabitol Dehydrogenase from Gluconobacter oxydans NH-10[J]. China Biotechnology, 2010, 30(11): 39-43

中图分类号： Q815

参考文献

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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