J. Gen. App!. Microbio!. Vol. 11, No. 1, 1965. A RACEMIZING ENZYME SYSTEM OF LACTOBACILL US PLANTAR UM TETSUO HIYAMA, SHOJI MIZUSHIMA and KAKUO KITAHARA Institute of Applied Microbiology, University of Tokyo, Tokyo ReceivedFebruary 15, 1965 It is well-known that the conversion of optically active lactate to the inactive form, racemization of lactate, can take place in some strains of bacteria such as the lactic acid bacteria and Clostridium sp. (1). In 1952, KITAHARAand coworkers obtained cell-free preparations of lactate racemase (trivial name " Recemiase," given by KATAGIRI and KITAHARA in 1937) from dried cells of Lactobacillus plantarum 11 which is one of the typical strains forming racemic lactate from glucose (2). Some enzymatic properties were reported with the partially purified " Racemiase " prepa- ration (3), and it was proved that nicotinamide adenine dinucleotide (NAD) is essential as a co-f actor to recover the activity with the preparation treated by active carbon or dialysis. It was difficult, however, to determine whether or not the racemization was catalyzed by a single enzyme because further purification was difficult due to its instability. In 1960, DENNIS and KAPLANdemonstrated the existence of NAD-depend- ent lactate dehydrogenases, each specific for the n(leavo)- or L(dextro)-isomer, in Lactobacillus plantarum ATCC 8041(4). It was proved in our laboratory that the formation of racemic lactate from glucose by resting cells of L. plantarum 11 is made possible by the cooperation of NAD-dependent D- and L-lactate dehydrogenases (5). This paper deals with the relationship between " Racemiase " and the lactate dehydrogenases in the organism, and discusses the mechanism of racemization. MATERIALS AND METHODS Materials Nicotinamide adenine dinucleotide (NAD) is a commercial product purchased from the Nutritional Biochemicals Corporation (Cleveland, Ohio). The reduced form of NAD was prepared by the method of RAFTER and COLOWICK(6), n- and L-lactic acids were prepared from the fermentation broths of Sporolactobacillus inulinus (7) and Lactobacillus casei TA, respec- tively. Lactic acid was neutralized by sodium or potassium hydroxide before use. Other materials were obtained commercially. Organism Lactobacillus plantarum 11 used in these experiments was from our own 51 52 HIYAMA, MIZUSHIMA and KITAHARA VOL. 11 culture collection and maintained on malt-bouillon agar. Cultivation of this organism was carried out exactly as described in the previous paper (5). Purification of lactate dehydrogenases As summarized in Table 1, the purification procedure was carried out according to the method described in the previous paper (5) with the following slight modification : for the D-enzyme, all buffers used throughout the procedure contained 0.01 M j3-mercaptoethanol to avoid loss of activity. D-Enzyme could be separated from L-enzyme by DEAE-cellulose chromato- Elution Volume (ml) Fig. 1. Elution patterns of crude "Racemiase" with DEAE-cellulose column. graphy (Fig. 1). It being difficult to separate the D-enzyme activity from the L-enzyme, the crude sonicate was heated at 50° for 3 minutes to inactivate the D-enzyme prior to the purification of the L-enzyme. For the details, see the previous report (5). Assays of enzyme activities The activity of lactate dehydrogenase was determined spectrophoto- metrically by the rate of NAD reduction or of NADH2 oxidation in the 1965 A Racemizing Enzyme System of Lactobacillus plantarum 53 Table 1. Purification procedures of D- and L-lactate dehydrogenases'. presence of lactate or pyruvate, respectively. Measurement was carried out at 340 mi and at 20° by the HITACHI EPU-2A spectrophotometer (Hitachi Ltd. Tokyo). Racemase activity was estimated by measuring the rate of D- or L-lactate formation from respective antipode. For the determination of optically active lactates, the manometrical method of KITAHARAand FUKUI (8 and 9) was used. When quick determination of racemase activity was required, D-lactate formed from L-isomer was calculated from the reduction rate of 2, 6-dichlorophenolindophenol with D-lactate dehydrogenase isolated from Lactobacillus casei (10). RESULTS Behavior of lactate dehydrogenases during purification of " racemiase ". The crude sonicate, which showed strong activity of lactate racemase in the presence of NAD, was fractionated by ammonium sulfate precipitation followed by column chromatographic fractionation with DEAE-cellulose. As summarized in Table 2, the second fractionation by DEAE-cellulose column chromatography caused an extreme loss of specific activity. Very active L-lactate dehydrogenase was detected in this fraction and only slight D-lactate dehydrogenase activity. The racemase activity was recovered by the addition of D-lactate dehydrogenase which had been purified separately. It is very likely that the loss of D-lactate dehydrogenase caused the inactivation of racemase activity due to less stability of the D-lactate dehydrogenase of this bacterium (5). Heat treatment, by which D-lactate dehydrogenase is inactivated (5), also caused inactivation of racemase activity. Fractionation of partially purified " Racemiase " by DEAE-cellulose column showed that the preparation contained NAD-dependent D- and L-lactate dehydrogenases (Fig. 1). Racemization of lactate by the mixture of dehydrogenases. The mixture 54 HIYAMA, MIzusHIMA and KITAHARA VOL. 11 Table 2. Purification of the racemase. Table 3. Activities of rasemase and dehydrogenases in crude sonicates. of n- and L-lactate dehydrogenases of L. plantarum, which were purified separately and had no racemase activity, could catalyze the racemization of lactate in the presence of NAD (Table 4). The data show that the rate of racemization, the n-lactate formation from L-lactate, nearly equals that of dehydrogenation, i.e., the pyruvate formation from L-lactate. Comparison of properties of "Racemiase " and dehydrogenases MICHAELIS'constants (Km) of L-lactate dehydrogenase for L-lactate and NAD were estimated by the method of LINEWEAVER-BURK'Splot. These 1965 A Racemizing Enzyme System of Lactobacillus plantarum 55 Table 4. Racemization by the mixture of two dehydrogenases. Table 5. Comparison of properties of "Racemiase " and L-dehydrogenase. values show good agreement with those of "Racemiase" reported by OBAYASHI et al. (3), using L-lactate as a substrate (Table 5). Racemase and dehydrogenase activities of crude extracts. The washed cells were suspended in 0.2 M-phosphate buffer, pH 7.0, and disrupted in a sonic disintegrator (Toyo-Riko, Ltd., Tokyo), 10-kc, 90-watt, for 30 minutes at 4°. The clear supernatant, obtained by centrifugation at 25,000 x g for 20 minutes, was used as a crude extract preparation. As shown in Table 3, the activity of the lactate dehydrogenase, from n-lactate and L-lactate to pyruvate, was 8.5 NM/mg cell/hr. Racemization, from L-lactate to n-lactate was 8.0 pM/mg cell/hr. This relationship between dehydrogenase and racemase activity was the same as found in the combined system of purified dehydrogenases shown in Table 4. The data show that the total activity of racemase comes from n- and L-lactate dehydrogenases in crude extracts which represents the total activity of the intact cell. The racemase activity of intact cells, however, was found to be several times lower than that of crude extracts. In the previous report it was shown that the backward reactions of n- and L-lactate dehydrogenases were inhibited by adenine nucleotides (5), as in the case of " Racemiase " reaction (3). It seemed reasonable, therefore, that the racemase activity was depressed in intact cells where concentrations of these substances were higher than in the 56 HIYAMA, MIZUSHIMA and KITAHARA VOL. 11 cell-free system. These observations were also supported by the fact that the activity of the lactate racemization in intact cells was further depressed under aerobic conditions where the intracellular concentration of pyruvate was increased by a lactate dehydrogenase-NADH2 oxidase system (15). Equilibria of dehydrogenase and racemase system. Equilibrium con- stants of D- and L-lactate dehvdrogenases were estimated from the concen- trations of substrates and products at the equilibrium points. The reaction was initiated from lactate and NAD. Formed NADH2 was determined spectrophotometrically Time (hr) Fig. 3. Time curves of reacemizing reaction. Time(min) Fig. 2. Equilibria of D- and L-lactate dehydrogenases. Reaction mixture is as follows: For D-reaction, 7.7 x 10-2 M D-lactate, 3.8 x 10-4 M NAD, D-lactate dehydrogenase (14~amoles/hr.) and 0.2M phosphate buffer, pH 7.2. For L-reaction, the same consti- tuents with exceptions of 7.7 x 10-2 M L-lactate and L-lactate dehydrogenase (16 p moles/hr.) (Fig. 2). From the results obtained the constants were calculated to be 2.4x 10.12 and 2.5 x 1012 at 20° for D- and L-lactate dehydrogenase, respec- tively. This explains the fact that the racemizing reaction in the present system reaches equilibrium where the D : L-ratio of lactate is 1:1, i .e., com- plete racemization. Figure 3 shows the time curves of the racemizing reaction by mixture of the dehydrogenases. The reactions were equilibrated at D : L=1:1 both from D- and L-isomers. The imbalance of the activities 1965 A Racemizing Enzyme System of Lactobacillus plantarum 57 Table 6. The activity of acetone dried cells. ATATI Fig. 4. Paper electrophoretic patterns of D- and L-lactate dehydrogenases, 0.01 M Tris-HC1 buffer containing 0.01 M jS-mercaptoe- of dehydrogenases (D: L=23 :14) has no influence on the final equilibrium point (D : L=1:1) as shown in Fig. 3. Other lactate dehydrogenases. As shown in Table 6, acetone treated cells do not show any dependency on NAD in the reduction of indophenol by D-lactate. The data suggest the presence of a D-lactate dehydrogenase which is independent of NAD and phrhaps has a flavin nucleotide as a prosthetic group. No further experiments to investigate this enzyme or to detect flavoprotein L-lactate dehydrogenases have been done.