The Journal of Biochemistry, Vol . 64, No. I, 1968

Purification and Properties of Lactate Racemase from Lactobacillus sake

By TETSUG HIYAMA, SAKUzo FUKUI and KAKUO KITAHARA*

(From the Institute of Applied Microbiology,University of Tokyo, Bunkyo-ku, Tokyo)

(Received for publication, February 12, 1968)

A lactate racemase [EC 5.1.2.1] was isolated and purified about

190 fold in specific activity from the sonic extract of Lactobacillus sake.

The purified preparation was almost homogeneous by ultracentrifugal and electrophoretical analyses. Characteristic properties of the enzyme were as follows : (a) absorption spectrum showed a single peak at 274 my,

(b) molecular weight was 25,000, (c) the enzyme did not require any additional cofactors and showed no activity of lactate dehydrogenase,

(d) K, values were 1.7 •~ 10-2 M and 8.0 •~ 10-2 M for D and L-lactate, respectively, (e) optimal pH was 5.8-6.2, (f) an equilibrium point was at a molar ratio of 1/1 (L-isomer/D-isomer), (g) the enzyme activity was

inhibited by atebrin, adenosine monosulfate, oxamate and some of Fe

chelating agents, (h) pyruvate and acrylate were not incorporated into lactate during the reaction, and (i) exchange reaction of hydrogen

between lactate and water did not occur during the reaction.

Since the first observation on the enzy the addition of both NAD and pyridoxamine matic racemization of optical active lactate phosphate. In this paper we deal with the by lactic acid bacteria had been reported by purification and properties of the lactate KATAGIRI and KITAHARA in 1936 (1), racemases racemizing enzyme from the cells of L. sake. for various substances were successively disco

vered in various organisms (2-8), and their MATERIALS AND METHODS physiological significance and reaction me Organism and Cultivation-Lacobacillus sake 012 chanism were discussed. which was isolated from sake starter in our laboratory On the biochemical racemization of lac was used. The bacterium was cultured at 30•Ž under tate, following two systems were presented : static condition in a medium containing 2 g of yeast one is lactate racemase [EC 5.1.2.1] in extract (Daigo Eiyo-Kagaku Co. Ltd., Osaka), lO g Clostridium (9, 10) and another is the coupl of Ehlrich's meat extract, 10 g of polypeptone (Daigo) ing system of NAD linked D and L-lactate and 15 g of glucose per liter. The medium was dehydrogenases [EC 1. 1.1.28 and 1. 1.1.27] in adjusted to pH 7.0 with KOH and sterilized at 120•Ž Lactobacillus (11). The former requires pyrido for 10 min. First seed culture was performed in 10 ml xamine-phosphate and ferrous ion as cofactors of the medium by 20 hr cultivation. To prepare and does not include any activity of lactate second seed culture, whole volume of the first seed dehydrogenase. culture was inoculated into 2 liters of the medium, and cultivation was carried out for 20 hr. Three Recently, we found that the racemase hundred milliters of the second seed culture was ino activity in Lactobacillus sake is not affected by culated into each of six Erlenmeyer flasks contain * Present address: Tokyo University of Agricul ing 10 liters of the medium per flask. The culture, ture, Tokyo. total volume of 60 liters, was incubated for 20 hr. 99 100 T . HIYAMA, S. FUKUI and K. KITAHARA

After the cultivation , the cells were harvested by hydrolyzed before use. Adenosine monosulfate (Na centrifugation and washed twice with 0 .5% NaCl and salt) was kindly supplied by Dr. M. Ishimoto, Universi finally with distilled water . The cells thus obtained ty of Hokkaido. were stored at -20•Ž . During the storage for a Other chemicals were commercial products. month any loss in the enzyme activity was not observed. From 60 liters culture , 12g of dry cells RESULTS were obtained . Purification of Lactate Racemase-All opera Assay of Lactate Racemase-Assay system for lactate racemase contained 10 ƒÊmoles of sodium D-lactate or tions were carried out at 4•Ž. Frozen cells, L-lactate, 20 ƒÊmoles of phosphate buffer 12 g dry weight, were suspended in deionized , pH 7.0, and racemase solution in a total volume of 0 .2 ml. The water to give a final volume of 200 ml. The reaction was carried out for 30 min at 30•Ž suspension was treated with sonic oscillation , then the mixture in a test tube was kept in boiling water for (10 kc, 100W) for 20 min to disrupt the cells. 30 sec to stop the reaction. An amount of optical Cell debris and intact cells were removed antipode formed from D or L-lactate was determined from the sonicated sample by centrifugation by the enzymatic methods of HIYAMA et al. (12) or at 25,000xg for 20 min. The supernatant was of HARN and BRUNS (13). The former is specific for incubated at 48•Ž for 3 min to precipitate D-lactate and the latter for L -lactate. One unit of heat-coagulable impurities. After removal of the enzyme is defined as an amount which con verts one ƒÊmole of L-lactate to D-isomer per hour precipitate by centrifugation, the supernatant was diluted with deionized water to 250 ml. under the conditions shown above. Specific activity To the diluted solution was added 62 g of is presented as an enzyme units per absorbance at 280mƒÊ. ammonium sulfate, and then the solution Determinaton of Metals-Metals in a racemase pre was allowed to stand for 2 hr. The precipitate

paration were analyzed by atomic absorption spectrum formed was removed by centrifugation. To technique using a Hitachi-Perkin Elmer atomic absorp the clear supernatant obtained, further addi

tion spectrophotometer. tion of 72 g of ammonium sulfate was per Separation of Lactic and Pyruvic Acids-Separation formed. The precipitate formed within 4 hr of lactic and pyruvic acids was carried out as follows : was collected by centrifugation at 10,000 •~ g a reaction mixture charged on a Dowex I •~ 8 (for for 10 min, and dissolved in deionized water mate type) column (I •~ 1 cm) was eluted successively of 21 ml. The solution was dialyzed overnight with 10 ml of 0.2 N and of 3.0 •~ formic acid. Lactic against one liter of deionized water, The and pyruvic acids were quantitatively recovered in dialyzate was kept at 50•Ž for 3 min. The the former and the latter eluates, respectively. By this procedure acrylic acid was not eluted from the precipitate formed by the treatment was re column. moved by ultracentrifugation at 105,000•~g Chemicals-D and L-lactic acids were prepared for 60 min. The supernatant was charged on from culture broths of Sporolactobacillusinulinus and a DEAE-cellulose column (3.5•~65 cm) equili Lactobacilluscasei, respectively. Lactic acid was ex brated with Mcllvaine buffer of 80 times tracted from the broth with ethyl ether. After dilution and pH 5.0. Elution was performed removal of ethyl ether from the extract , the acid with the same buffer containing 0.13m KCl was diluted with small amount of water and decolored at an elution rate of 7.0 ml per minute. The by passing through an activated carbon bed. The enzyme was recovered in tubes of number lactic acid was turned into zinc lactate by treatment 9-13 (each tube 200 ml). The enzyme solu with zinc carbonate. Zinc lactate was recrystallized twice from water. Purified free acid was prepared tion (1000 ml) was concentrated by dialysis against flakes of polyethylenglycol (Tpye 6000 from the lactate by passing through a cation ex , D changer resin column. Lactic acid obtained was ainippon Seiyaku Co. Ltd., Osaka) for 10 hr. neutralized with NaOH and then used as substrate The concentrated sample (10 ml) was diluted for lactate racemase. with deionized water to decrease ionic strength . Pyruvic acid (3-14C) and tritiated water were Then, the sample was applied to a second purchased from the Radiochemical Centre, England. column chromatography of DEAE-cellulose

Acrylate methyl ester (2, 3-14C) was obtained from (1 •~ 15 cm) which was pretreated with the Nuclear Research Chemicals, Inc., U.S.A., which was same buffer of McIlvaine mentioned above . Lactate Racemase 101

TABLE I Purification of lactate racemase.

FIG. 1. Sedimentation patterns of lactate racemase. Ultracentrifugation was performed with the

purified enzyme dissolved in 0.5m KC1, pH 5.0, in a Hitachi ultracentrifuge. Protein concentra tion was 1.7 mg per ml. Experimental conditions were as follows : temperature, 18•Ž; speed, 59,400 rpm ; bar angle, 55ß; pictures on the left and right were obtained after 28 and 48 min of centrifugation, respectively.

Elution was also carried out in the same manner as presented above. Fractions giving high specific activity (tube number 7-13, each tube 10 ml) were collected and dialyzed against deionized water for 4 hr. The dialyzed preparation (120 ml) was charged on a DEAE FIG. 2. Electrophoretical pattern of lactate cellulose column (1 •~ 2 cm) buffered with the racemase. same concentration of Mcllvaine buffer in Electrophoretical pattern was obtained by the above, then the enzyme adsorbed was disc electrophoresis under the conditions of eluted with 2.0 ml of 0.5 Nt KCl. The enzyme ORNSTEIN and DAVIS (14) preparation obtained was concentrated 190 . The final preparation was used in the follow fold in specific activity. Whole procedure of ing sections for enzymatic study. the purification is summarized in Table I. 102 T. HIYAMA, S. FUKUI and K. KITAHARA

Patterns in Ultracentrifugation and Electro molecular weight 12,600, Sankyo Co. Ltd., phoresis-Asedimentation pattern of the enzyme Tokyo) bovine serum albumin (assigned M.W., is shown in Fig. 1, in which one major and 67,000, Sigma Chemicals Co., U.S.A.) and one minor peaks are observed. The major r-globulin (assigned M.W., 160,000, Sigma) component (4.2 S) was isolated by separa were used as the standard. A molecular tion cell in ultracentrifugation and was found weight of 25,000 was obtained from the plots to have a higher specific activity than that of elution volumes versus logarithm of mo of the original sample. The major peak, lecular weight (Fig. 3). therefore, was considered to be lactate race Optimal pH-pH dependency curve of the mase. An analysis by disc electrophoresis racemase is shown in Fig. 4, in which an under the conditions used by ORNSTEINand optimal pH range of 5.8-6.2 is presented in DAVIS(14) gave a single band of protein as McIlvaine buffer. is presented in Fig. 2. Activities of Lactate Dehydrogenases-By ap Absorption Spectrum-Absorption spectrum plying both methods of NAD reduction (16) of the enzyme showed only one peak at and of 2, 6-dichloroindophenol reduction (17), 274 mƒÊ in a wave lenghth range of 240 to it was found that the enzyme preparation did 700 mƒÊ, and no other peak was detected at not include any activities of lactate dehydro an enzyme concentration of 1.7 in absorbance genases, such as NAD or flavin dependent L at 274 mƒÊ. Any change in absorption spectrum and D-lactate dehydrogenases. does not occur by the addition of substrate, such as D or L-lactate.

Molecular Weight-Molecular weight was

determined by the gel-filtration method of ANDREWS (15). Yeast cytochrome c (assigned

FIG. 3. Estimation of molecular weight by

gel-filtration. 0.1 ml of the purified sample was applied to a Sephadex G-200 column (1.3 •~ 32 cm) equilibrated

with Mcllvatine buffer, twice the concentration FIG. 4. Optimal pH. and pH 5.1, and elution was performed by the Buffers used were as follows : same buffer. Elution volume was estimated by -•›- McIlvaine buffer. absorbance at 280 mƒÊ for albumin and r-globulin, -•œ- 0.1 M Phosphate buffer at final concentra

by absorbance at 450 mƒÊ for cytochrome c and by tion. enzymatic activity for the racemase. -•¢- 0 .1 M Acetate buffer at final concentration Lactate Racemase 103

Fin. 5. Effect of dilution. The reaction mixture contained 10 ƒÊmoles of FIG. 6. Time course of racemization. D-lactate or L-lactate, 20 pmoles of acetate buffer , The reaction was performed in the presence

pH 5.7, and purified enzyme in 0.2 ml. The of L-lactate under the conditions shown in Fig. 5. reaction was carried out for 30 min at 30•Ž, and -•›-Lactate formed.

then optical antipode formed from active lactate -•œ- L-Lactate remained . was analyzed. The enzyme activity was plotted versus the amount of enzyme.

-•›- Substrate is n-lactate.

-•œ- Substrate is L-lactate .

Effect of Enzyme Concentration-Proportion ality'of the activity to enzyme concentration isjshown in Fig. 5. When a molecular weight

of 25,000 was used, turnover numbers of the

enzyme at 30•Ž in 0.1 M acetate buffer, pH

5.7, were calculated to be 1000 and 400 moles

per mole enzyme per minute for D and L lactate, respectively. E7uilibrium and Stoichiometry Fig. 6 shows FIG. 7. Lineweaver-Burk's polts. a time course of the racemase reaction using Reaction mixture contained 20 ƒÊmoles of L-lactate as substrate. The reaction reached acetate buffer, pH 5.7, 1 unit of the racemase an equilibrium at a point of equimolar con and substrate in 0.2 ml. ƒÒ is reaction velosity,

centration Of D and L-isomers. Quantitative ƒÊ mole per 30 min.

,conversion of L-lactate to D-lactate can also -•›- Substrate is D-lactate. be observed in Fig. 6. -•œ- Substrate is L-lactate.

Michaelis Constants-From Lineweaver cations, such as Mn++ Fe++ Mg++ Cu++, Burk's plots, respective Km values were calcu Co++, Zn++, and Ca++ in chloride form, also lated to be 1.7 •~ 10-2 M and 8 •~ 10-2 M for D

- and L-lactate (Fig. 7). gave no effect at a concentration of 5 •~ 10-5 M. Inhibitors-The effect of various inhibitors Cofactors-Several compounds known as on the reaction is summarized in Table ‡U. cofactors were added to the reaction mixture The compounds in column A of the Table at zero time of incubation to examine their were added to the reaction mixture at zero effects on the activity. Additions of NAD, time of incubation, and compounds in column NADP, FAD, FMN, pyridoxal phosphate, B were preincubated for 30 min with the and pyridoxamine phosphate showed no ef enzyme at 0•Ž. The reaction was started by fect at a concentration of 1.0 •~ 10-4 M. Divalent 104 T. HIYAMA, S. FUKUI and K. KITAHARA

TABLE ‡U

Effect of inhibitors. Reaction mixture contained 6.6pmoles of L-lactate, 13.2pmoles of acetate buffer, pH 5.7, 2 units of the enzyme, and inhibitor in a volume of 0.2 ml. The reaction was performed for 30 min at 30•Ž, and then n-lactate formed was determined.

1) Some of inhibitors used here inhibit lactate dehydrogenase in an assay system for n-lactate, so that the correction was made for the effect of the inhibitor on the enzymatic determination of n-lactate. 2) Without preincubation with inhibitor. 3) D-Lactate was used as substrate. 4) With preincubation with inhibitor. Chelators for iron ions were used as inhibitor. Pretreatment with chelator was carried out for 30min at 0•Ž before the reaction. The reaction was started by the addition of L-lactate. EDTA, 8-hydroxyquinoline, ƒ¿, ƒ¿•Œ-dipyridyl, and ƒÍ-phenanthroline are chelators for ferrous ion; Tiron (pyrocatechol-3, 5-disulfonate Na), and Nitroso-R-salts (ƒ¿-nitroso-ƒÀ-naphtol-3, 6-disulfonate Na) for ferric ion. the addition of L-lactate. Cyanide and some racemase reaction was carried out in the chelators for iron ion have inhibitory effect. presence of radioactive pyruvate (3-14C) to This finding suggests that iron in the enzyme investigate the reaction mechanism. The has some relation to the activity. Atebrin, reaction mixture contained 0.25 ƒÊmoles of adenosine monosulfate (18 ), and oxamate labeled pyruvate (approximately 104 cpm),

(19) were also found to be strong inhibitors. 25 ƒÊmoles of sodium L-lactate, 50 ƒÊmoles of Metals in Bound Form-Approximately 4 ƒÊg acetate buffer, pH 5.7, and 2 units of the of iron in 1 mg of the enzyme was determin purified enzyme in a total volume of 0.45 ml. ed by the method of atomic absorption As a comparative system, the sonic extract spectrum. When the molecular weight is of Lactobacillus plantarum, which was a racemiz assumed to be 25,000 for the enzyme, the ing system consisting of NAD linked D and iron content obtained here corresponds to L-lactate dehydrogenases, was used with a two atoms of iron in one molecule of the supplement of NAD of 1.5 pmoles. The reac enzyme. The presence of both copper and tion was carried out for 10 hr at 20•Ž. After manganese was demonstrable, but their con the reaction, 10 ƒÊmoles of cold pyruvate was tents were less than 0.4 ƒÊg per mg enzyme. added to the reaction mixture, and then Incorporation of Pyrurate into Lactate-The lactic and pyruvic acids in the mixture were Lactate Racemase 105

TABLE ‡V

Incorporation of labeled pyruuate into lactate.

Reaction conditions were described in the text.

fractionized by Dowex-1 •~ 8 column chromato poration of acrylate into lactate was not graphy. Each fraction of lactic and pyruvic observed. acids was dried on a planchet and the Incorporation of Tritium of Tritiated Water radioactivity was determined by a Geiger into Lactate-To examine an exchange reaction Miiller counter. As can be seen in Table ‡V between hydrogen at a position of a or ƒÀ-carbon in lactate molecule and hydrogen , significant radioactivity was not detected in the lactate fraction obtained from the reac in water during the racemization, the reac tion mixture containing the racemase of L. sake, tion was performed in tritiated water. The while in the reaction mixture of L. plantarum reaction mixture contained 10 )ymoles of sodi system almost all of radioactivity given as um L-lactate, 20 ƒÊmoles of acetate buffer, pyruvate was recovered in the lactate fraction. pH 5.7, and 2 units of the enzyme in tritiated Incorporation of Acrylate into Lactate-The water (50 mCi) of 0.3 ml. The reaction was racemization reaction in the presence of label carried out for 3 hr at 30•Ž. After the reac

ed acrylate (2, 3-14C) in stead of pyruvate was tion, cold lactate of 50 ƒÊmoles was added to carried out by the same manner as the case the reaction mixture, then lactate was fraction

of incorporation test of pyruvate. With the ized from the mixture by the column chro

reaction mixture of the racemase, any incor matography of Dowex-l •~ 8. Lactate fraction

TABLE ‡W

Incorporation of tritium in tritiated water into lactate.

Reaction was carried out as described in the text.

1) Calculation was performed from the initial rate of enzyme reaction by applying the calculation basis shown in the text, because reaction did not yet reach an equilibrium state after 3 hr incubation. 2) Enzyme activity was so high that the hydrogen exchange reaction between lactate and water must have reached an equilibrium state after 3 hr incubation. Therefore, specific radioactivities of lactate should have been equal to those of water. This is the calculation basis for this reaction. 106 T. HIVAMA. S. FUKUI and K. KITAHARA

was collected in a vial for scintillation count and D-lactate must be observed. The race ing, and lyophilized. The lyophilized sample mase prepared here, however, did not show was dissolved in 5 ml of redistilled water and any activity of lactate dehydrogenase. This lyophilized again. Lyophilization was repeat finding is certified by the observation show ed three times to remove radioactive water ing no incorporation of pyruvate into lactate. completely. The final sample was suspended Therefore, this mechanism should be elimi in a Hyamine-Water-Dioxane system for liquid nated. The possibility of the mechanism of scintillation measurement (20). Radioactivity internal hydride shift, which was recently was determined by a Packard Tri-Carb model proposed for the racemase system of Clostridium 314AX liquid scintillation counter. As refer butylicum (21), was not examined in this paper,

ence systems, lactate dehydrogenase systems, because we could not find a testing system such as the sonic extract of L. plantarum and for this mechanism. crystalline L-lactate dehydrogenase from pig Consequently, we have at present no idea

(Bohringer and Sohne, Germany), were used for the explanation of the reaction mechanism with supplemet of 2 ƒÊmoles of NAD. Results of lactate racemase prepared from L. sake. are summarized in Table ‡W, in which hypo Physiological situation of the racemase in

thetical radioactivities are also listed. The lactic acid fermentation of L. sake will be hypothetical values are obtained under the discussed below. Lactate racemase could be assumption that the hydrogen exchange reac extracted by sonic treatment in a correspond

tion takes place at a rate of one atom per ing activity to the activity shown by the

one atom between tritiated water and lactate intact cells. Activity of NAD dependent L

as the racemase or dehydrogenase reaction lactate dehydrogenase was detected in the

proceeds. By comparing the experimental sonic extract, and NAD dependent incorpora values with the hypothetical values, it is con tion of pyruvate into lactate was observed by cluded that significant amount of incorpora the cells treated with cationic detergent (un tion of tritium of water into lactate does not published data). When the racemase forma occur. tion was repressed by the addition of sodium acetate to a growth medium (22), the bacte DISCUSSION rium produced only L-lactate. It is, therefore,

For the enzymatic racemization of lactate, the case of L. sake that L-lactate once formed

the following three mechanisms have been via glycolytic pathway is converted to taken into consideration : (a) Dehydration, (b) DL-form by the racemase, in contrast to the Dehydrogenation and (c) Internal Hydride case of L. plantarum, in which both D and

Shift. We will discuss what mechanism of L-lactate are formed directly from pyruvate

these three will be acceptable for the race at the same rate (11). Distribution of lac mase obtained here. tate racemase in lactic acid bacteria, which If dehydration mechanism, which was were DL-lactate forming strains, was examined.

proposed for the racemase of Clostridium aceto All strains tested, such as L. japonicus, L. brevis, butylicum (9), is correct, following observations Pediococcuslindneri, Pc. hennebergi,required NAD should be obtained: (1) the exchange reaction essentially for the activity of racemization.

between hydrogen at ƒ¿ or ƒÀ-carbon in lactate Thus it seemed that a racemization system and hydrogen of water occurs as the racemiza consisting of D and L-lactate dehydrogenases tion proceeds, and (2) the conversion of acrv was more common in lactic acid bacteria late, one of dehydrated compounds of lactate, than the racemase discovered in L. sake. to lactate takes place. However, both were The authors wish to thank Kyowa Hakko Co., Ltd. not observed. Thus, the dehydration mecha for a large scale cultivation of the bacterium. nism should be eliminated. If dehydrogena tion mechanism, which was presented for the REFERENCES Lacobacillus plantarum's system (11, 19 ), is cor (1) H. Katagiri and K. Kitahara, J. Agr. Chem. rect, dehydrogenase activities toward both L Soc. Japan, 12, 844 (1936) Lactate Racemase 107

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