Gluconobacter oxydans木糖醇脱氢酶基因的克隆表达及木糖醇的转化分析

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.coli JM109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 h 28.0 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。

Cloning, Expression and Conversion Analysis of the Xylitol Dehydrogenase from Gluconobacter oxydans

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 K_m value for NADH was 57.8mmol/L and the V_(max) was 1209. lmmol/( 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.