Purification and properties of malolactic enzyme from Lactobacillus murinus CNRZ 313

The malolactic enzyme of Lactobacillus murinus was purified 79 fold. Mr = 220,000 as determined by gel filtration and gradient gel electrophoresis. The enzyme consists of two apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulfate. NAD protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. The apparent Km's for malate, NAD, and Mn2+ were 2.31 X 10(-2), 4.5 X 10(-4), and 1.4 X 10(-4) mM, respectively. Maximum enzymatic activity was observed at 37 degrees C and pH 5.5 in 0.2 M phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8000 and 16,200 cal mol-1 for the temperature values more than and less than 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD and manganese-dependent reaction L-malate----L-lactate + CO2. Therefore, this enzyme can be distinguished from the well-known malic enzymes [L-malate: NAD+ oxidoreductase, oxaloacetate decarboxylating (EC 1.1.1.38) or decarboxylating (EC 1.1.1.39)].