Malolactic Enzyme of Lactobacillus plantarum PURIFICATION, PROPERTIES, AND DISTRIBUTION AMONG BACTERIA*

m e malolactic enzyme of Lactobacillus plantarum was purified from 5.6 units/mg to a specific activity of 266 units/mg of protein, The enzyme has an isoelectric point of pH 4.4. The molecular weight is M, = 140,000 as determined by gradient gel electrophoresis. “he en- zyme consists of two probably identical subunits (Mr = 70,000) that were observed after treatment with so- dium dodecyl sulfate. Malolactic enzyme catalyzes the NAD- and manganese-dependent reaction tmalate 4 COz + L-lactate. Therefore, this enzyme can be distin-guished from the well known malic enzymes (L-malate: NAD’ oxidoreductase, oxalacetate-decarboxylating Malolactic enzyme is found in most lactic acid bacteria (Lactobacteriaceae); it has not been detected in other bacteria. in one 4.4. dialysis, 3.4 of protein with a specific activity of malolactic enzyme 265 units/mg of protein were

The list of the International Enzyme Commission (1) contains the following "malic enzymes" that are specific for Lmalate: 1) L-malate:NAD' oxidoreductase (oxalacetate-decarboxylating), EC 1.1.1.38, first observed in Lactobacillus arabinosus (synonymous with Lactobacillusplantarum, the now accepted name (2); 2) L-malate:NAD' oxidoreductase (decar-boxylating}, EC 1.1.1.39, isolated from Ascaris lumbricoides (3) and from Streptococcus faecalis (4), this enzyme does not decarboxylate oxalacetate; 3) L-mdate:NADP' oxidoreductase (oxalacetate-decarboxylating}, EC 1.1.1.40, originally isolated from pigeon liver (5). The activity of the malic enzymes EC 1.1.1.39 and EC 1.1.1.40 can be determined either by measuring spectrophotometrically the formation of NAD(P)H or by measuring the formation of carbon dioxide. However, the activity of the enzyme from the lactic acid bacterium L. plantarum (synonym for L. arabinosus) can only be determined by the measurement of carbon dioxide. Neither NADH nor pyruvate is found as an end product of the reaction of malate that yields L-lactate and CO2 only (2). The occurrence of lactate instead of pyruvate has been originally regarded as the result of the joint action of a malic enzyme and a constitutive L-lactate dehydrogenase in L. arubinosus (=L. plantarurn), because this latter enzyme could not be separated from the malate decarboxylating activity during its partial purification (6, 7).
Later on it was shown that L-lactate dehydrogenases are not involved in the formation of L-lactate from L-malate by L. plantarum (8). The enzyme involved in the reaction was shown to have a molecular weight of about 150,000. Recently, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ TO whom correspondence should be addressed. a malolactic enzyme from Leuconostoc mesenteroides was partially purified (9), with a molecular weight of 235,000.
This paper describes the puriEication of the malolactic enzyme of L. plantarum. Some characteristics of this enzyme are determined. Of particular interest was obtaining information about the occurrence of malolactic enzyme among lactic acid bacteria and related bacteria.

Materials
Phenyl-Sepharose CL 4B, Sephadex  (synonym for L. plantarum). All bacterial strains were from the collection of this institute. The strains were originally obtained from various sources. We have to thank all colleagues who supplied these strains.

Preparation of Cell-free Extracts
The cells of L. plantarum were grown in Leuconostoc oenos medium (DSM, Catalogue of strains 1977: casein peptone, tryptic digest, 10 g; yeast extract, 5 g; glucose, 10 g; fructose, 5 g; MgSO,. 7H20 = 0.2 g; MnSOI.HzO = 0.05 g; diammonium citrate, 3.5 g; Tween 80, I d, tomato juice, Fdtered 100 d, cysteine-HCI, 0.5 g; distilled water, 900 ml), modified by the addition of 10 g of L-malic acid, final pH 5.5-6.0. Three liters of medium in a 5-liter flask were sterilized by steaming for 30 min, inoculated with 1% of a preculture, and incubated with slight agitation at 30 "C for 14 h. The cells (about 15 g) were washed, suspended in 0.01 M phosphate buffer, pH 6.0, containing 0.03% sodium azide, and macerated for 2.5 min in the C02cooled cell homogenizer (MSK, Braun, Melsungen) at 4,000 rpm (2.5 mI of buffer and 0.5 g of gIass beads, diameter 0.11-12 mm, per g of cells, fresh weight). Supernatant and rinsing liquid were centrifuged at 10,OOO X g for 30 min to remove cells and debris. The cell-free extract (91.6 ml) contained 19.1 mg of protein and 105 units of malolactic enzyme per ml.
Purification of Malolactic Enzyme from L. plantarum All procedures were carried out at 4 "C.
Step I : Precipitation with Protamine Sulfate-The cell-free extract was treated with protamine sulfate (1 ml of 1% (w/v) solution per 100 mg of protein).
Step 2: Ion Exchange Chromatography-One-third of the enzyme preparation after Step 1 (310 mg of total protein) was loaded onto a TEAE 23-cellulose column (diameter, 26 mm; volume, 40 ml; prepared from 6 g of TEAE-cellulose in 0.01 M phosphate buffer, pH 6). washed with phosphate buffer, and eluted (4.7 ml cm-2 h") with a linear The abbreviations used are: TEAE, triethylarninoethyl; SDS, sodium dodecyl sulfate. gradient of KC1 (0-0.5 M) over a 360-ml volume phosphate buffer. Fractions of 6 ml were collected. The highest specific activity ofmalic enzyme was observed at the conductivity of the eluant of 8 mS (see Fig. 1).
Step 3: Hydrophobic Chromatography-The most active fractions obtained from Step 2 were dialyzed with a PM 10 ultrafilter (Amicon), adjusted to 1 M (NH4)?S04, and applied to a column of phenyl-Sepharose 4B (diameter, 15 mm; gel volume, 8 m l ; equilibrated with 1 M ammonium sulfate in 0.01 M phosphate buffer, pH 6, and 0.03% sodium azide). Then the column was washed with equilibrating solution. For elution (22 ml cm? h"), a decreasing gradient of 1 to 0 M ammonium sulfate was used. The highest specific activity of malolactic enzyme observed in one fraction was 211 units (see Fig. 2).
Step 4: Preparative Isoelectric Focusing-The three preparations of malolactic enzyme from Step 3 obtained by separate purifications were combined and diluted with bidistilled water to 106 ml. The sample was stirred and 6 ml of Ampholine pH 4-6 were added. To this solution, 5 g of Sephadex IEF were added and this mixture was left standing overnight for soaking. Then the gel was degassed and poured on the glass plates (215 X 215 mm) to prepare the gel flat bed (thickness, 2 mm). After focusing for 16 h at 40 Watts in the flat bed apparatus (PHARMACIA FBE 3,000), the gel was cut into 13 strips of 15-mm breadth each, then the gel strips were transferred into test tubes. Before measuring the pH value, 2 to 3 ml of water were added. Protein was eluted by washing the gels (placed in glass columns 15 X 150 mm) with 25 ml of 0.5 M KC1 in 0.01 M phosphate buffer, pH 6. Each eluate was dialyzed twice with 50 ml of 0.01 M phosphate buffer, pH 6 , and concentrated with ultrafilter membranes (PM 10). Malolactic enzyme was observed in one fraction at pH 4.4. After dialysis, 3.4 mg of protein with a specific activity of malolactic enzyme of 265 units/mg of protein were recovered.

Polyacrylamide Gradient Gel Electrophoresis
The molecular weight of malolactic enzyme was estimated by polyacrylamide gradient gel electrophoresis with PHARMACIA PAA 4/30 gradient gel and the flat bed apparatus (PHARMACIA FBE 3,000). Before applying the samples, the gels were equilibrated for 20 min at 70 V. 0.68 mg of lyophilized protein was dissolved in 100 pl of electrode buffer (0.09 M Tris, 0.08 M boric acid, 0.93 g/liter of NA2EDTA, pH 8.4). Ten pl of this solution was loaded onto the gel. Electrophoresis lasted 16 h at 150 V. A mixture of proteins (HMW Calibration kit) was run simultaneously (fixing and staining, 15 h in 0.02% Coomassie blue in 7% acetic acid; destaining, 24 h in 7% acetic acid).

SDS-Polyacrylamide Gradient Gel Electrophoresis
The procedure used was similar to the method of Weber and Osborn (11). The same equipment and staining procedures as for gradient gel electrophoresis were employed. 0.68 mg of lyophilized protein was dissolved in 100 pl of electrode buffer (0.04 M Tris, 0.02 M sodium acetate, pH 7.4, 2 mM EDTA, 0.2% (w/v) sodium dodecyl sulfate). To 50 pl of this solution, 5% (v/v) mercaptoethanol was added and the mixture was immersed in boiling water for 5 min. Ten pl of the protein samples were applied to the gradient plates.

Enzyme Assay
The activity of malolactic enzyme was determined by a modified method of Kaufmann et al.

Determination of Cell Weight and Protein
The weight of the cells (fresh weight) was determined by weighing the sediment after centrifugation at 6,000 X g for 15 min. The protein content of the extracts was determined by the biuret reaction or spectrophotometrically at 280 and 260 nm according to Warburg and Christian (17).

Measurement of Conductivity
The conductivity (recorded as miIliSiemens) of the fractions after chromatography was measured with the conductivity meter CDM 2B of Radiometer, Copenhagen.

Purification of Malolactic Enzyme-For the purification
of malolactic enzyme, the lactic acid bacterium L. plantarum B38 was used. This strain is very similar to the strain used by Korkes and Ochoa (2) in their paper describing "malic enzyme." L. plantarum B38 shows a particular high activity of malolactic enzyme when grown at inducing conditions (8).
Malolactic enzyme was purified 48-fold from a cell-free extract of L. plantarum with a specific activity of 5.5 units/ mg. The purification of malolactic enzyme from L. plantarum is summarized in Table I (

for details see "Experimental Procedures''). A preparation with a specific activity of 265 units/ mg was obtained after isoelectric focusing.
The isoelectric point was pH 4.4. Polyacrylamide gradient gel electrophoresis and staining with Coomassie blue showed that this sample of malolactic enzyme yielded one protein band (Fig. 3A). By comparison with the known molecular weights of the "kit"proteins, the malolactic enzyme showed a molecular weight of about M, = 140,000 (Fig. 4).
After treatment with SDS to separate malolactic enzyme into polypeptide subunits, gradient gel electrophoresis revealed only one protein band by staining with Coomassie blue (see Fig. 33). According to its RF value and by comparison with the "calibration kit" proteins, the molecular weight of the polypeptide subunit from malolactic enzyme was about M, = 70,000 (see Fig. 5). This is about one-half of the molecular weight of the entire malolactic enzyme. Therefore, it can be assumed that malolactic enzyme consists of two subunits that are apparently (but not necessarily) identical. " After protamine sulfate precipitation, the enzyme preparation was divided into three equal parts that were purified separately. This table combines the results of these purifications.
'All three preparations.
. Reactions of Malolactic Enzyme-Using a purified preparation of malolactic enzyme, the previously described (8) stoichiometry of the reaction of L-malate + Cop + L-lactate was c o n f i e d , by enzymatic determination of malate and lactate and manometric determination of COZ (5 pmol of Lmalate consumed yielded 4.8 pmol of L-lactate and 5.5 pmol of Cop). When purified malolactic enzyme (1.4 units) was used in the usual spectrophotometric tests (366 nm), no activity (i.e. less than 0.1%) of malate dehydrogenase (substrate oxalacetate and NADH2), malic enzyme (substrate L-malate and NAD), or lactate dehydrogenase (substrate pyruvate and NADHp) was detected. Oxalacetate is decarboxylated to pyruvate at pH 6 a t a similar rate as malate.
Substrate Affinity of Malolactic Enzyme-The substrate affinities for L-malate, NAD, and Mn'+ ions of malolactic enzyme of L. plantarum were determined with an enzyme preparation with a specific activity of 297 units/mg. Except for the substrate tested, the composition of the test system was the same as in the enzyme assay. From the Lineweaver-Burk plots, the following K,,, values were obtained: L-malate, K,,, = 9.5 mM; NAD, K, = 59-mM; Mn", Distribution of Malolactic Enzyme in Bacteria-Among 59 strains of lactic acid bacteria belonging to 25 species of the genera Lactobacillus, Leuconostoc, Pediococcus, and Strep-  tococcus, only 6 strains did not contain malolactic enzyme (see Table 11). These "negative" strains belonged to four species, other strains of these species (except S. faecalis) contained the enzyme. A further investigation of several Gram-positive bacteria that may be regarded to show Some relatedness to lactic acid bacteria did not detect malolactic enzyme. This enzyme was not found in strains ofthe following species: Propionibacterium acidipropionicum, Propionibacterium thoenii, Aerococcus uiridans, Bifidobacterium bifidum, Sporolactobacillus inulinus, Peptococcus uariabilis, Peptostreptococcus anaerobius, Eubacterium limosum.

DISCUSSION
The malolactic enzyme from L. plantarum described in this paper is identical with the enzyme from L. arabinosus (synonym of L. plantarum) that was reported by Korkes et al. (2) and called malic enzyme by these authors. In contrast to the first reported malic enzyme from pigeon liver (EC 1.1.1.40) that yields Con, pyruvate, and reduced coenzyme from Lmalate and NADP (5), the end products of the reaction of malolactic enzyme from L. plantarum are COz and L-lactate. Therefore, malolactic enzyme is different from the malic enzyme of pigeon liver (EC 1.1.1.40) and also from the other true malic enzyme (EC 1.1.1.39) that has been found in S. faecalis and Lactobacillus casei (4).
In this paper, the molecular weight of malolactic enzyme from L. plantarum as determined by gradient gel electrophoresis was found to be about M, = 140,000. The malolactic enzyme of LC. mesenteroides that was partially purified by Lonvaud-Funel and Strasser de Saad The treatment of malolactic enzyme with sodium dodecyl sulfate led to the separation of the enzyme into its subunits: gradient gel electrophoresis revealed only one band that showed a molecular weight of 70,000. Therefore, it is assumed that malolactic enzyme consists of two probably identical subunits.
Alizade and Simon (12) and Kraus et al. (13) have investigated the fermentation of labeled L-malate by intact cells of LC. mesenteroides. Their results led to the assumption that a multienzyme complex catalyzes the steps L-malate + oxalacetate -+ pyruvate -+ L-lactate. It was further assumed that the intermediary compounds oxalacetate and pyruvate are not liberated from the enzyme complex. The direct decarboxylation of L-malate to L-lactate was regarded as rather unlikely.
The results of our paper do not support the hypothesis that malolactic enzyme is an aggregation of several different enzymes. Its two subunits are apparently identical. Therefore, if malolactic enzyme is regarded as a multienzyme complex, then the components must be covalently linked for they cannot be separated by sodium dodecyl sulfate.
The necessity of NAD ( K , = 59 mM) for the reaction of malolactic enzyme is a typical characteristic of an oxidoreductase. However, no reduction of NAD can be observed by the usual spectrophotometric methods. If not the activating compound but the end products of the reaction of malolactic enzyme are being considered, then this enzyme could be regarded as a carboxylyase. In this case, the original designation L-malate:NAD+ oxidoreductase (oxalacetate-decarboxylating) (EC 1.1.1.38) could be transferred to the malic enzyme (sensu stricto) of Schizosaccharomyces pombe, that reacts with L-malate and NAD, yields pyruvate and NADH, and decarboxylates oxalacetate (14).
The reaction catalyzed by malolactic enzyme that is very frequent in lactic acid bacteria (but is limited to this group of organisms) does not yield metabolizable end products, reduction equivalents, or energy equivalents. The significance of malolactic enzyme that many lactic acid bacteria contain in high activities probably rests in the compensation of the increase in hydrogen ions that are formed during the fermentation of carbohydrates (15,16). This decrease of acidity caused by malolactic enzyme (the dicarboxylic acid is converted to the monocarboxylic lactic acid) is a selective advantage for lactic acid bacteria that occur in environments with a high sugar content in the presence of malic acid. The malolactic fermentation of wine is of considerable importance for the wine industry and the malolactic enzyme is the key enzyme in this process.