大肠杆菌苹果酸脱氢酶基因mdh的克隆、高效表达及酶学性质

Cloning,Expression,and Characterization of a Malate Dehydrogenase Gene from Escherichia coli

以大肠杆菌基因组DNA为模板,扩增得到苹果酸脱氢酶（mdh）编码基因mdh,构建了重组菌pET-28a-mdh/BL21并成功表达了mdh,大小约36 000。选用Ni柱亲和层析法纯化具有活性的苹果酸脱氢酶（mdh）,纯化后比酶活达到112.5 U/mg,纯化倍数达2.62倍,回收率为59%。并对该酶的酶学性质进行了初步研究,其中反应最适pH值为6.0,在pH值2.0~6.0范围内稳定;反应最适温度为37℃,在42℃以下酶的稳定性较好。K＋对酶有明显的激活作用,Cu2＋对酶有抑制作用,Hg2＋和Zn2＋对酶有很强的抑制作用。醇类对酶的活力影响不大,丙三醇可显著提高酶的热稳定性。酶动力学参数以草酰乙酸为底物的Km为0.235 mmol/L,Vmax为0.47μmol/（L.min）。

Malate dehydrogenase（MDH） gene was amplified via PCR from the chromosome of Escherichia coli in this manuscript.The PCR product was cloned into the expression vector pET-28a（＋）.The resulted recombinant plasmid was transformed into E.coli BL21（DE3）.Induced by 0.5 mmol/L IPTG,MDH,a 36KDa protein,was successfully expressed in E.coli BL21（DE3）.An active MDH was purified by Ni-NTA column affinity Chromatography,with the specific activity of 112.5 U/mg,the purification multiple of 2.62,and the recovery rate of 59%.In a preliminary study,the enzymatic properties of the purified His-tagged enzyme were characterized.It was found to have pH and temperature optima of 37 ℃ and 6.0,respectively.The enzyme was stable when pH and temperature kept in the range of 2.0 to 6.0 and blow 42 ℃,respectively.Its activity was activated by K＋ dramatically,inhibited by Cu2＋,seriously inhibited by Zn2＋ and Hg2＋.Although alcohols have little effect on this enzyme,glycerol could dramatically improve the thermal stability of MDH.When oxaloacetic acid was used as substrate,the enzyme kinetic constants of Km and Vmax was 0.235 mmol/L and 0.47 μmol/（L·min）,respectively.