Cloning, Expression And Characterization Of A Malate Dehydrogenase Gene From Escherichia Coli

Malate dehydrogenase (MDH) is the key enzyme of tricarboxylic acid cycle (TCA), which is commonly observed in living organisms. MDH catalyses the reversible oxidation-reduction of malate and NAD+ to oxaloacetate and NADH.One target fragment about 939 bp was amplified from Escherichia coli genomic DNA via PCR and cloned into the expression vector pET-28a (+). The recombinant plasmid pET-28a-mdh was transformed into E. coli BL21 (DE3) and then induced to express a fussion protein by IPTG. The purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to Sepharose. The specific activity of coarse enzyme is 43 U/mg. After purification via affinity chromatography with Ni2+, the specific activity of MDH is 112.5 U/mg, the purification multiple is 2.62, the recovery is 59%. We study preliminarily on the characterization of the purified enzyme. The optimum activity of MDH is observed at 37℃and pH 6.0, the range of temperature stability is blow 42℃, the range of pH stability is from 2.0 to 6.0, the activity of enzyme is activated by K+ dramatically, inhibited by Cu2+, Zn2+ and Hg2+ inhibited the activity strongly. Alcohols have little effect on the specific activity of the enzyme. Glycerols can improve the thermal stability of MDH dramatically. The enzyme kinetic constants (Km and Vmax) are as follows:using oxaloacetic acid as substrate, Km is 0.235 mmol/L, Vmax is 0.47μmol/L.min.In this research, mdh was cloned into expression vector pET-22b (+) and the recombinant plasmid pET-22b-mdh was transformed into BL21 (DE3). Positive transformant was cultivated and IPTG was added into culture to induce secreted expression of MDH. One-step extraction using lytic buffer is used to get the protein in periplasm of Escherichia coli, the protein is MDH whose specific activity is 48.2 U/mg.Based on the characterization of MDH, the thermal stability of MDH is relatively poor. Site-directed mutagenesis is performed so that the thermal stability of MDH can be improved. using the recombined plasmid pET-28a-mdh as template, lover lap extension PCR is used to introduce Q229E,L225K,D45E mutations into mdh. The thermolysin mutants were cloned to pET-28a (+) vector and expressed, purified, and characterized for their activities and thermal stabilities. The relative activity of Q229E mutant is 73% after keeping warm at 45℃for half an hour and 44% at 65℃. While the relative activity of L225K mutant and D45E mutant is 56.5% and 38.6% respectively after keeping warm at 45℃, the loss of relative activity of the two mutants at 65℃is much, the relative activity is 28.6% and 9% respectively; The relative activity of orginal MDH is 45.6% at 45℃and 3.7% at 65℃after keeping warm for half one hour. So we describe the Q229E mutant has higher thermal stability than the original MDH and the two mutants L225K and D45E have less influence on the thermal stability, but the optimum temperature of all is still 37℃, the specific activity of MDH has few changes.