克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究

以克雷伯氏菌基因组DNA为模板，扩增得到编码甘油脱氢酶（GDH）的基因dhaD，将其克隆到大肠杆菌表达载体pET-28a(+)上，在E.coliBL21（DE3）中诱导表达，利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH)，纯化后比酶活达到156U/mg，纯化倍数达4.6倍，回收率为67.4%。并初步研究了该酶的酶学性质，酶反应的最适pH为11.0，在pH7.0～12.0范围内稳定；酶反应的最适温度为30℃，稳定范围为25～45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。

Expression, Purification and Enzymatic Characterization of Klebsiella sp. glycerol dehydrogenase in E. coli

The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified. and was inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21 (DE3). SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the results showed a single band about 39kDa on SDS-PAGE gel, and the specified activity was about 156U/mg. The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0. The optimum reactive temperature was 30℃，and the GDH was more stable on the temperature of 25℃～45℃. The Km value was 0.54mmol/L and Vmax was 0.49 μmol/(mL·min) in the glycerol.