Please use this identifier to cite or link to this item:
|Scopus||Web of Science®||Altmetric|
|Title:||Malolactic enzyme from Oenococcus oeni: Heterologous expression in Escherichia coli and biochemical characterization|
del Hierro, A.
|Citation:||Bioengineered Bugs, 2013; 4(3):147-152|
|Christina Schümann, Herbert Michlmayr, Andrés M. del Hierro, Klaus D. Kulbe, Vladimir Jiranek, Reinhard Eder and Thu-Ha Nguyen|
|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.|
|Keywords:||Escherichia coli; Malates; Lactic Acid; Malate Dehydrogenase; Bacterial Proteins; Enzyme Stability; Gene Expression; Kinetics; Molecular Weight; Oenococcus|
|Rights:||© 2013 Landes Bioscience|
|Appears in Collections:||Agriculture, Food and Wine publications|
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.