Malolactic enzyme from Oenococcus oeni: Heterologous expression in Escherichia coli and biochemical characterization

Schümann, Christina, Michlmayr, Herbert, del Hierro, Andrés M., Kulbe, Klaus D., Jiranek, Vladimir, Eder, Reinhard and Nguyen, Thu Ha (2013) Malolactic enzyme from Oenococcus oeni: Heterologous expression in Escherichia coli and biochemical characterization. Bioengineered, 4 (3). (doi:10.4161/bioe.22988).

Abstract

Malolactic enzymes (MLE) are known to directly convert L-malic acid into L-lactic acid with a catalytical requirement of nicotinamide adenine dinucleotide (NAD⁺) and Mn²⁺; however, the reaction mechanism is still unclear. To study a MLE, the structural gene from Oenococcus oeni strain DSM 20255 was heterologously expressed in Escherichia coli, yielding 22.9 kU l^-1 fermentation broth. After affinity chromatography and removal of apparently inactive protein by precipitation, purified recombinant MLE had a specific activity of 280 U mg^-1 protein with a recovery of approximately 61%. The enzyme appears to be a homodimer with a molecular mass of 128 kDa consisting of two 64 kDa subunits. Characterization of the recombinant enzyme showed optimum activity at pH 6.0 and 45°C, and K_m, V_max and k_cat values of 4.9 mM, 427 U mg^-1 and 456 sec^-1 for L-malic acid, 91.4 μM, 295 U mg^-1 and 315 sec^-1 for NAD⁺ and 4.6 μM, 229 U mg^-1 and 244 sec^-1 for Mn²⁺, respectively. The recombinant MLE retained 95% of its activity after 3 mo at room temperature and 7 mo at 4°C. When using pyruvic acid as substrate, the enzyme showed the conversion of pyruvic acid with detectable L-lactate dehydrogenase (L-LDH) activity and oxidation of NADH. This interesting observation might explain that MLE catalyzes a redox reaction and hence, the requirements for NAD⁺ and Mn²⁺ during the conversion of L-malic to L-lactic acid.

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Contributors

Author: Vladimir Jiranek

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