Preparation, Enzymatic Properties And Application Of Malate Dehydrogenase From Porcine Heart

Malate dehydrogenase (EC 1.1.1.37, MDH) is one of the key enzymes in TCA cycle, it reversibly catalyzes the convertion of malic acid and oxaloacetate. Widely existing in animal organs, bacteria and plants, MDH has been used in the assay of organic acids such as L-malic acid, acetate acid and citric acid, even for early diagnosis of disease. The research on the extraction, purification, enzymatic properties and stabilities of MDH originated from porcine are introduced. And the application of MDH on the assay of L-malic acid in wine was also cited.The extraction and purification of MDH from porcine were perfomed. MDH was extracted by tissue crushing, ammonium sulfate duel-precipitation, and purified through DEAE Sepharose F.F, Phenyl Sepharose 6 F. F. hydrophobic chromatography and Sephadex G-75 Fine Gel. The final specific activity of MDH was 1212.97 U/mg, and 122.64-fold concentration was obtained with enzymatic activity recovery of 53.64%. A single MDH protein band was shown on SDS-PAGE gel finally.Enzymatic properties and stabilities of extracted MDH from porcine was studied. Results shows that the optimal temperature and pH of MDH from porcine was 50℃and 8.0 respectively. MDH was relatively stable under the temperature below 50℃, but the enzyme activity decreased quickly at the temperature above 60℃. Meanwhile, the enzyme activity is relatively stable in the pH range from 7.0 to 9.0, shows slightly alkali-resistant. It was found that the molecular weight of MDH was 69 kDa which included two subunits with the molecular weight of each about 34 kDa. The Km of MDH versus substrate oxaloacetate was 84.6μmol/L. From our experiments, MDH was strong inhibited by metal ions such as Ag+, Hg2+ and Zn2+, whereas K+ had significant activated effect and Ca2+ and Mg2+ had little effect on MDH. Moreover, the stability of MDH could be improved by glycerol.Based on the experiments above, the primary application of MDH was studied. new method was exploited for assaying L-malic acid in wine by MDH and GOT dual-enzymatic system. The assay was linear over L-malic acid of 0.005 to 0.3 g/L at the temperature of 25℃and detective wavelength at 340 nm.It was proved to be simply and accurately applicable to L-malic acid determination in wine by the experiment of precision and recovery (93.2%~104%).