【摘要】 从氧化葡萄糖酸杆菌(Gluconobacter oxydans)的基因组DNA上扩增出木糖醇脱氢酶基因xdh,构建了诱导型表达载体pSE-xdh,导入E.coliJM109后获得了高效表达木糖醇脱氢酶基因的重组菌JM109/pSE-xdh。通过HisTrap HP亲和层析和SephacrylS-300分子筛两步纯化从细胞中得到纯酶,并对酶学性质进行研究。XDH最适还原反应的pH值为5.0,最适还原反应的温度为35℃;最适氧化反应的pH值为11.0,最适氧化反应的温度为30℃。重组菌中的XDH依赖NADH,对NADH的米氏常数Km=57.8 mmol/L,最大反应速率Vmax=1209.1 mmol/(ml.min)。重组菌的XDH酶活力为13.9 U/mg。利用重组菌和原始菌混合静止细胞转化D-木酮糖,16 h28.0 g/L D-木酮糖生成16.7 g/L木糖醇,而原始菌单独转化只生成8.3 g/L木糖醇。更多还原

【Abstract】 Xdh encoding xylitol dehydrogenase(XDH)gene was amplified from the genome DNA of G.oxydans NH-10.The recombinant plasmid pSE-xdh was constructed by inserting xdh genes into expression vector pSE380 and transformed into E.coli JM109.The recombined XDH was purified through two steps including HisTrap HP affinity chromatography and SephacrylS-300 gel filtration chromatography,and then the enzymatic properties were investigated.The optimum pH and temperature for reduction conditions of XDH were 5.0 and 35℃,while that for the oxidation conditions were pH 11.0 and 30℃.XDH was a kind of NADH-dependent dehydrogenase,and the Km value for NADH was 57.8mmol/L and the Vmax was 1209.1mmol/(ml·min).The XDH activity of recombinant strain was 13.9 U/mg.16.7 g/L xylitol was obtained from 28.0 g/L D-xylulose in 16 h by mixed fermentation of resting cells which was composed of original strain and recombinant strain,whereas control strain produced 8.3 g/L xylitol.These results demonstrated that increasing XDH activity could improve xylitol productivity.更多还原