Expression, Purification and Functional Characterization of Two Recombinant Malate Dehydrogenases from Mortierella isabellina

Abstract

To study the characteristics of two malate dehydrogenases (MDHs), their coding genes MIMDH1 and MIMDH2 were cloned and expressed in Escherichiacoli BL21 cells. The molecular weights of both recombinant enzymes partially purified using Ni-NTA affinity chromatography were 32 kDa, and the specific activities of purified MIMDH1 and MIMDH2 were 329.3 and 241.3 U/mg protein, respectively. The optimal temperatures for MIMDH1 and MIMDH2 activities were 55 and 30^oC, respectively, with 70% MIMDH1 activity and ≥70% MIMDH2 activity remaining after 30 min incubation at 45^oC. Addition of 2 mM Zn²⁺ enhanced MIMDH1 activity, whereas the addition of other metal ions resulted in different degrees of inhibition. The inhibitory effect of Co²⁺ was most pronounced on MIMDH1, reaching 82.8% inhibition. Addition of 2 mM Ba²⁺ or Mn²⁺ increased MIMDH2 activity, the addition of other metal ions resulted in different degrees of inhibition. Furthermore, MIMDH1 was stable at pH range of 7.5–8.5, with optimal activity observed at pH of 8.0. MIMDH2 showed similar stability at the same pH range, but was optimal at pH of 7.5. The V_max and K_M values for recombinant MIMDH1 and MIMDH2 catalyzing the reduction of oxaloacetate to malate were 17.66 µmol mg^–1min^–1 and 0.541 mM, 15.59 µmol mg^–1min^–1 and 0.683 mM, respectively.