作　　者： (封志媚）; (赵雅童）; (刘业学）; (路福平）; (李玉）;

机构地区： 天津科技大学生物工程学院工业微生物教育部重点实验室,天津300457

出　　处： 《生物技术通报》 2017年第8期186-191,共6页

摘　　要： 对NADPH依赖的甘露醇脱氢酶的异源表达和对果糖的转化情况进行分析,为在细胞内构建甘露醇的合成途径奠定基础。构建重组菌株BL21(DE3)/p ET28a-mdh,对其进行诱导发酵后,通过His标签对目的蛋白进行纯化,并利用HPLC分析纯化后的甘露醇脱氢酶转化果糖生成甘露醇的情况。成功构建了NADPH依赖的甘露醇脱氢酶重组表达菌株BL21(DE3)/p ET28amdh,并对表达后的甘露醇脱氢酶进行了纯化,测得纯化后的甘露醇脱氢酶酶活力为270 U/m L。对酶法转化果糖得到甘露醇的转化条件进行优化,确定最佳的转化条件为:当底物浓度为300 g/L,反应初始p H5.8,温度40℃,NADPH终浓度为9 mmol/L时,甘露醇转化率可以达到97.4%。实现了NADPH依赖的甘露醇脱氢酶在大肠杆菌中的活性表达,为进一步研究大肠杆菌合成甘露醇的代谢调控提供了依据。 This work is to analyze the heterogeneous expression of NADPH-dependent mannitol dehydrogenase and the transformationof fructose for laying a foundation of constructing mannitol synthetic way. The recombinant strain BL21（DE3）/p ET28a-mdh of expressingmannitol dehydrogenase was constructed and induced,and the target protein was purified with His-tag. Mannitol production from mannitoldehydrogenase transforming fructose was analyzed by HPLC. As results,the recombinant strain was constructed successfully and the mannitoldehydrogenase was purified. The activity of the purified mannitol dehydrogenase was 270 U/m L. The conditions of fructose transforming tomannitol by purified mannitol dehydrogenase were optimized,and the optimal conditions were as follows ：300 g/L substrate fructose,p H5.8,9 mmol/L NADPH and incubation at 40℃. Under the optimized conditions,the transformation rate of mannitol reached 97.4%. In conclusion,the NADPH-dependent mannitol dehydrogenase was expressed successfully in Escherichia coli,providing the basis for studying the metabolismregulation of mannitol synthesis in E. coli.

关 键 词： 甘露醇脱氢酶 甘露醇 大肠杆菌 纯化