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)。

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.