title: Malolactic enzyme from Oenococcus oeni: heterologous expression in Escherichia coli and biochemical characterization.
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 Mn ( 2+) ; 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 Km, Vmax and kcat 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 Mn ( 2+) , 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 Mn ( 2+) during the conversion of L-malic to L-lactic acid.
authors.affiliation: Department of Food Sciences and Technology, Division of Food Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Schümann
authors.forename: Herbert
authors.initials: H
authors.lastname: Michlmayr
authors.forename: Andrés M
authors.initials: AM
authors.lastname: Del Hierro
authors.forename: Klaus D
authors.initials: KD
authors.lastname: Kulbe
authors.forename: Vladimir
authors.initials: V
authors.lastname: Jiranek
authors.forename: Reinhard
authors.initials: R
authors.lastname: Eder
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008970:Molecular Weight; D056585:Oenococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008293:Malates; D019344:Lactic Acid; C030298:malic acid; C037731:malolactic enzyme; D008291:Malate Dehydrogenase