Abstract: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.
