Production of Racemic Lactic Acid in Pediococcus Cerevisiae Cultures by Two Lactate Dehydrogenases

JOURNAL OF BACrERIOLOGY, Feb. 1975, p. 600-607 Vol. 121, No. 2 Copyright 0 1975 American Society for Microbiology Printed in U.SA.

Production of Racemic Lactic Acid in Pediococcus cerevisiae Cultures by Two Lactate Dehydrogenases

GEOFFREY L. GORDON AND H. W. DOELLE* Downloaded from Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia Received for publication 22 November 1974 Nicotinamide adenine dinucleotide (NAD)-dependent D(-)- and L(+)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for D(-)-lactate dehydro- http://jb.asm.org/ genase and 136,000 to 139,000 for L(+)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by D(-)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, L(+)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis- Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with on September 19, 2016 by University of Queensland Library increased affinity for both substrates at the acid pH. a-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevistae.

By definition, all lactic acid bacteria produce racemic lactate by Pediococcus lindneri and P. some lactic acid during fermentation of car- hennebergi is brought about by the presence of bohydrates. In most cases this lactic acid is two different lactate dehydrogenases within either levorotatory or dextrorotatory, but occa- their cells. The present investigation was under- sionally a mixture of both isomers can be taken in an attempt to provide information at formed. Lactobacillus sake has been found to the enzyme level on the mechanism of produc- form L( +)-lactic acid by the action of a nicotin- tion of racemic lactate mixtures in cultures of P. amide adenine dinucleotide (NAD)-dependent cerevisiae. L( + )-lactate dehydrogenase (EC 1.1.1.27; L+LDH), which in the presence of lactate MATERIALS AND METHODS racemase (EC 5.1.2.1) results in the production of a racemic mixture of lactic acid in the culture Materials. All cofactors, substrates, nucleotides, glycolytic intermediates, as well as dithiothreitol (14). A similar situation has also been reported (DTT) and tris(hydroxymethyl)aminomethane recently in L. curvatus (20). However, certain (Tris), were purchased from Sigma Chemical Co. (St. other members of this group of organisms pro- Louis, Mo.). Lactate dehydrogenases from rabbit duce racemic mixtures of lactic acid by the muscle and L. leichmannii were purchased from simultaneous action of NAD-dependent D(-)- Boehringer-Mannheim GmbH (Mannheim, Ger- lactate dehydrogenase (EC 1.1.1.28; D-LDH) many). Diethylaminoethyl-Sephadex A-25 and Seph- as well as L+LDH (4, 5, 10, 17). arose 6B were products of Pharmacia Fine Chemical Since pediococci are traditionally regarded as Company (Uppsala, Sweden); hydroxylapatite Bio organisms that produce racemic lactic acid as a Gel HTP was obtained from Bio-Rad Laboratories major end product of glucose (Richmond, Ca.). Ammonium sulfate was special catabolism (1), the enzyme grade from Schwarz/Mann (Orangeburg, question arises: how is this racemic mixture N.Y.). All other chemicals were analytical grade. actually formed? Preliminary studies by Organisms and culture conditions. P. cerevisiae Hiyama et al. (14) indicated that production of 813 and P. pentosaceus 9206 were obtained from the 60( VOL. 121, 1975 LACTATE DEHYDROGENASES FROM P. CEREVISIAE 601 National Collection of Dairy Organisms (National units were expressed as micromoles of NADH pro- Institute for Research in Dairying, Shinfield, Read- duced or oxidized per minute at 30 C. Specific activity ing, Berkshire, England) and from the National is defined as units per milligram of enzyme protein. Collection of Industrial Bacteria (Torry Research The assay system used for lactate racemase and the Station, Aberdeen, Scotland), respectively. Both were defimition for lactate racemizing units of activity were maintained by monthly subculture on MRS agar (3) those used by Hiyama et al. (14).

slopes, which were stored at 4 C after growth for 24 h Analytical methods. Residual glucose in the me- Downloaded from at 28 C. dium was determined by a mixed enzyme-dye method Large-scale growth of P. cerevisiae was undertaken (A. St. G. Huggett and D. A. Nixon, Biochem. J. 66: in a 10-liter carboy of MRS medium containing only 12P, 1957). 1.0% (wt/vol) glucose. The complete medium without Isomer production of lactic acid in culture superna- glucose was sterilized by autoclaving at 115 C for 20 tants, as well as in assays of lactate racemase, was min; a 40% glucose solution, sterilized separately by determined enzymatically by the method of Hohorst autoclaving, was then added aseptically to a final (15), using L+LDH from rabbit muscle and D-LDH concentration of 1.0%. Bacterial growth in the carboy from L. leichmannii. was initiated with a 1% (vol/vol) inoculum of a 24-h Dry weights of the cells were determined in pre- culture grown in the same medium. The culture was weighed glass vials after addition of a washed-cell http://jb.asm.org/ incubated at 28 C for 18 h, with constant but slow suspension and drying at 105 C to constant weight. magnetic stirring. The cells were harvested by centrif- Protein concentration in cell-free extracts was mea- ugation, washed with cold 0.9% (wt/vol) NaCl, and sured by the biuret method (12), with crystalline stored as a frozen cell paste. bovine serum albumin as standard, whereas protein Cells for lactate racemase studies were grown in concentrations of all other purification steps were 100-ml amounts of modified MRS, which was pre- estimated by using their absorbance at 260 and 280 pared by omitting sodium acetate and ammonium nm (21). citrate and by reducing the glucose concentration to Molecular weight estimations. Native molecular 0.1%. weights were estimated by gel filtration through a on September 19, 2016 by University of Queensland Library Preparation of cell-free extracts. Ten grams of Sephadex G-200 column (2.6 by 65 cm) eluted with thawed cell paste was suspended in about 200 ml of 0.05 M sodium phosphate buffer (pH 7.0) containing 2 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT. Standard proteins and methods for their mM DTT and 2 mM sodium-DL-lactate, and dis- assay have been described previously (11). rupted in 50-ml fractions with 50 g of glass beads The native molecular weight of D-LDH was esti- (0.10- to 0.11-mm diameter) at 4,000 rpm in a Braun mated electrophoretically, by the method of Hedrick cell homogenizer (type MSK; Melsungen, Germany) and Smith (13), in an alkaline polyacrylamide gel for 15 min. During disruption, the glass vessel was system (2) as described previously (11). However, an cooled by a continuous stream of liquid CO2. The estimate for L+LDH was not possible in this system opaque, straw-colored liquid was decanted and then owing to the extreme lability of this enzyme at clarified by centrifugation at 37,000 x g for 30 min. alkaline pH. Therefore, the native molecular weight of The resultant supernatant was then used for all L+LDH was estimated electrophoretically at pH 7.0 enzymatic procedures. by the procedure of Weber and Osborn (22) modified Enzyme assays. Both lactate dehydrogenases were by the omission of sodium dodecyl sulfate. Five assayed spectrophotometrically at 340 nm by using a polyacrylamide gel concentrations (5 to 9%). were used Pye-Unicam SP 8000 recording spectrophotometer for the estimation by the method of Hedrick and with 3.0-ml, 1.0-cm path length quartz cuvettes. Smith (13). Lactate oxidation was measured in a system contain- Subunit molecular weights of the two enzymes were ing 25 mM Tris-maleate buffer (pH 8.2), 2.0 mM estimated in gels containing 0.1% sodium dodecyl sodium NAD, and 10 mM lithium L(+)- or 10 mM sulfate by the method of Weber and Osborn (22) as D(-)-lactate in a final volume of 3.0 ml. Pyruvate described previously (11). reduction was measured in a 3.0-ml system containing 25 mM Tris-maleate buffer (pH 5.4 or 7.4) and RESULTS concentrations of sodium pyruvate and sodium reduced NAD, as indicated in Table 1. Dehydrogenase Growth characteristics and lactate production by P. cerevisiae. The growth charac- TABLE 1. Summary of assay conditions for pyruvate teristics ofP. cerevisiae grown in standing batch reduction in P. cerevisiae culture at 28 C in a 10-liter carboy are shown in Fig. 1. A cell yield of 4 g (dry weight per liter) Sodium pyr- Sodium W- Assay pH uvate (mM) NADH (mM) corresponds to about 7 g (wet weight) of cells/ liter. The cells were harvested early in the sta- 5.4 2.0 0.04 tionary phase after 18 h of growth, as lactate 7.4 10 0.1 production appeared to have ceased by this stage. 5.4 0.3 0.02 During the fermentation, L( + )-lactic acid was 7.4 1.0 0.02 the first isomer to appear in the culture me- 602 GORDON AND DOELLE J. BACTERIOL. sodium phosphate buffer (pH 7.0) containing 2 mM DTT. This solution was layered onto a Sepharose 6 6B column (2.8 by 70 cm), which had been equilibrated with the same buffer, and the TL was eluted at a flow rate of 30 ml/h.

column Downloaded from L+LDH was eluted first and was followed closely by D-LDH, with a slight overlap of activities. Fractions with more than 50% of the activity of each peak fraction were pooled (Fig. 2). I- The first peak was concentrated to about 5 ml _HoursI0 in an Amicon ultrafiltration cell fitted with a FIG. 1. Growth characteristics of P. cerevisiae UM-50 membrane. The concentrated solution NCDO 813 in MRS broth (3) at 28 C. Symbols: 0, was diluted to 50 ml with 0.05 M sodium http://jb.asm.org/ bacterial dry weight; 0, L(+)-lactate; 0, D(-)-lac- phosphate buffer (pH 6.0) containing 2 mM tate; V, culture pH; A, glucose in medium. DTT and reconcentrated to about 5 ml. This solution was then applied to an hydroxylapatite dium, whereas the D(-) isomer did not appear column (1.0 by 30 cm), which had been equili- until several hours later when the pH had fallen brated with the same buffer. The column was to about 5. At this point, further L(+) acid eluted at a flow rate of 3.6 ml/h with a 200-ml production ceased. M sodium phos- Lactate racemng activity in cell-free ex- linear gradient of 0.05 to 0.5

phate buffer (pH 6.0). The single activity peak on September 19, 2016 by University of Queensland Library tracts of pediococci. Cell-free extracts pre- fractions were pooled and concentrated by ul- pared from cells of P. cerevisiae and P. trafiltration (UM-50 membrane), diluted with pentosaceus, grown in the absence of acetate 0.05 M sodium phosphate buffer (pH 7.0) con- and citrate, exhibited considerable lactate ra- taining 2 mM DTT, and reconcentrated as cemizing activities of 4.1 and 3.6 U/mg of protein, respectively. However, after overnight Sepharose 65 dialysis of the extracts against the buffer system rI I used for cell-free extract preparation, this activity was completely lost, while specific activities of both lactate dehydrogenases were essentially constant. Partial purification of NAD-dependent L+LDH and D-LDH from P. cerevisiae. En- zyme activities were monitored by lactate oxidation throughout the purification procedure. All operations were carried out between 0 and 4 C, except the ammonium sulfate fractionation, which was performed at room temperature (about 20 C). After centrifugation at 4 C, the cell-free extract was equilibrated at room temperature, after which it was adjusted to 45% ammonium sulfate saturation by the slow addition of the solid chemical (277 g/liter) together with slow magnetic stirring while a constant, gentle stream of dry nitrogen was played over the liquid surface. The pellet formed after centrifugation (20 min at 37,000 x g) was discarded, whereas the ammonium sulfate concentration of the supernatant was increased to 70% saturation (by addition of 171 g of solid chemical per liter to the 45% saturated-ammonium sulfate Elution Volume (ml) supernatant, with identical precautions). Cen- FIG. 2. Elution profiles of D-LDH and L+LDH trifugation (20 min at 37,000 x g) produced a activity during purification. Symbols: *, L+LDH; 0, pellet which was suspended in 5 ml of 0.05 M D-LDH; A, absorbancy at 280 nm. VOL. 121, 1975 LACTATE DEHYDROGENASES FROM P. CEREVISIAE 603 before. Sufficient glycerol was added to the L+LDH and D-LDH from P. cerevisiae. Max- partially purified L+LDH preparation to give imum activity for L+LDH was found at pH 7.6, final concentration of 30% (vol/vol), with the whereas that for D-LDH was at pH 9.6. preparation then being stored at 4 C without Pyruvate reduction by D-LDH demonstrated appreciable loss of activity over a period of an activity peak at pH 8.0 (Fig. 3), which several months. to a at shifted sharp peak pH 3.6 when the Downloaded from The second peak eluted from the Sepharose pyruvate concentration was lowered from 5.0 to 6B column was concentrated, diluted with 0.05 0.5 mM. On the other hand, pyruvate reduction M Tris-hydrochloride buffer (pH 8.0) contain- by L+LDH exhibited maximum activity in the ing 2 mM DTT and 0.05 M NaCl, and reconcen- pH range 4.5 to 6.0 at either pyruvate concentrated to about 5 ml. This preparation was then tration. applied to diethylaminoethyl-Sephadex A-25 In 1963 it was estimated that the internal pH column (2.0 by 30 cm), which had been equili- of actively metabolizing cells of L. plantarum is brated with the same buffer. The column was 5.4 (19). This value, as well as the pH value

eluted at a flow rate of 100 ml/h with a 500-ml normally regarded as physiological (7.4), was http://jb.asm.org/ linear gradient of 0.05 to 0.5 M NaCl. The single used in an estimation of the reversibility of both activity peak fractions were pooled and concen- enzymes. Reaction velocities for lactate oxida- trated by ultrafiltration (UM-50 membrane), tion and pyruvate reduction of both enzymes at diluted with 0.05 M Tris-maleate buffer (pH these pH values were estimated from the pH 8.0) containing 2 mM DTT, reconcentrated, profiles for lactate oxidation and those shown in and stored frozen without appreciable loss of Fig. 3. From this data it was calculated that D- LDH activity over a period of several lactate oxidation was 6.1% and 1.1% of pyruvate

months. reduction at pH 7.4 and 5.4, respectively, for on September 19, 2016 by University of Queensland Library A summary of a typical purification proce- D-LDH and 2.7% at pH 7.4 for L+LDH. dure appears in Table 2; representative elution L+LDH was not reversible at pH 5.4. profiles are displayed in Fig. 2. Both enzyme Michaelis constants for substrates. Mi- preparations were examined for homogeneity by chaelis constants (Ki) for all substrates of both polyacrylamide gel electrophoresis, but in both enzymes are given in Table 3. Values for lactate cases the preparations possessed a major pro- and NAD were estimated for the purified en- tein band, which corresponded to the activity zymes from linear double-reciprocal plots at band, contaminated with two or three other one substrate concentration. Michaelis con- faint protein bands. stants for pyruvate and NADH were estimated pH optima and reversibility of the reac- at pH 5.4 and 7.4 by plotting saturation curves tions. Hydrogen ion concentration was found to at four concentrations of the second substrate. have different effects on lactate oxidation by The reciprocals for apparent maximum veloci-

TABLE 2. Summary of the purification of lactate dehydrogenases from P. cerevisiae Total L+LDH D-LDH Ttlnucleic t Purification step Vol (ml) protein acid Sp act Total Yield Sp act To Yield l(mg) activity (%) |(mg) (U/mg) (U) (U/mg) activ(ty(U) 1. Cell-free extract 168 1008 722 0.0257 25.97 100 0.0177 17.86 100

2. Ammonium sulfate fraction- 5.0 285 119 0.0610 17.39 67 0.0457 13.04 73 ation, 45 to 70% saturation 3. Sepharose 6B chromatography: Peak I 18.5 77.7 4.3 0.2093 16.27 63 0.0127 0.99 6 PeakII 18.0 41.4 3.1 0.0294 1.22 5 0.3067 12.70 71 4. Hydroxylapatite chromatogra- 16.5 3.9 0.1 1.8116 7.17 28 0 0 O phy of peak I at pH 6.0 5. DEAE-Sephadex chromatog- 27.0 2.2 0 0 0 0 1.5700 3.39 19 raphy of peak II at pH 8.0a - DEAE, Diethylaminoethyl. 604 GORDON AND DOELLE J. BACTERIOL. ties for the four different second substrate concentrations were then plotted against the reciprocal of the substrate concentration to give the Km of the second substrate (Fig. 4). All E substrate saturation curves for bo'th enzymes TO were hyperbolic, double-reciprocal plots which Downloaded from were linear and gave Km values for both substrates which increased with increasing pH. Effect of pyruvate analogues. Of the four analogues tested (a-ketobutyric, oxamic, oxalic, http://jb.asm.org/

40-

20 O-

E on September 19, 2016 by University of Queensland Library

-8 4 8 '/Pyruvote (mM'4) 'C 0 FIG. 4. Example for determination of Km value for

60- ~ substrates of pyruvate reduction. The reaction mixtures contained 25 mM Tris-maleate buffer (pH 5.4), partially purified D-LDH (2.7 ,gg of protein), NADH as indicated, and the following concentrations of A, A, 80- pyruvate: 0.1 mM; 0.2 mM; 0, 0.3 mM; *, 0.5 mM.

20~~~~ pH and malonic acids), only a-ketobutyric could be used for the oxidation of NADH. The Km values 2 4 6 8 at pH 5.4 and 7.4 were 22 and 30 mM, respec- citrate phosphate tris maleate tively, for D-LDH and 32 and 86 mM, respectively, for L+LDH. FIG. 3. Effect of pH on D-LDH and L+LDH ac- When the same analogues were tested as tivities. The enzyme reactions were assayed by pyru- inhibitors of pyruvate reduction, only 5.0 mM vate reduction with 25 mM buffer, 0.1 mM NADH oxamatt caused 50% inhibition of D-LDH at and either 0.5 mM (A) or 5.0 mM pyruvate (B). Sym- pH bols: 0 and A, L+LDH; 0 and A, D-LDH. 5.4, whereas 0.5 mM oxamate and 0.5 mM oxalate at pH 5.4 and 7.0 mM oxamate at pH TABLE 3. Michaelis constants (K.) for substrates of 7.4 resulted in 50% inhibition of L+LDH. L+LDH and D-LDH Differential properties of the enzymes. While studying the lactate dehydrogenases of L. Km (mM substrate) plantarum, Dennis and Kaplan (4) found sev- Variable substrate pH L+LDH D-LDH eral chemical procedures that initiated mark- edly different responses from the D-LDH and Pyruvate 5.4 1.0 0.15 the L+LDH of this organism. First, lactate 7.4 8.0 0.67 oxidation by L+LDH was severely inhibited by 8 mM oxamate, whereas D-LDH was not af- NADH 5.4 0.02 0.0083 fected at all by the equivalent oxamate concen- 7.4 0.04 0.01 tration. Second, the D-LDH was totally inacti- D(-)- or L(+)-lactate 8.2 67 30 vated by incubation at 50 C for 3 min, whereas L+LDH was not denatured until a temperature NAD 8.2 5.0 1.05 of 80 C was reached. However, in the present study both enzymes from P. cerevisiae were VOL. 121, 1975 LACTATE DEHYDROGENASES FROM P. CEREVISIAE 605 inactivated by incubation at 55 C for 3 min, and nondenaturing polyacrylamide gels, and by 8 mM oxamate was not inhibitory to lactate electrophoresis in polyacrylamide gels contain- oxidation by either enzyme. ing sodium dodecyl sulfate (Table 5). Figure 5 is Regulatory properties. Many glycolytic and the calibration curve for estimation of the hexosemonophosphate pathway intermediates, native molecular weights by electrophoresis in as well as D(-)- and L(+)-lactate, were tested neutral polyacrylamide gels. Other calibration Downloaded from on pyruvate reduction at both pH values by curves have already been published (11). both enzymes, and not one had any effect. On the other hand, adenosine triphosphate, adeno- DISCUSSION sine diphosphate, guanosine triphosphate, and A previous study of the production of D(-)- NAD inhibited both enzymes to varying degrees and L(+)-lactic acid in cultures of P. cerevisiae (Table 4). Adenosine triphosphate appears to be has shown that the ratio of L(+) acid to total the most potent inhibitor, and its action was lactic acid is high initially, but decreases as the found to be competitive with respect to NADH cultures grow (9). This finding was verified http://jb.asm.org/ and noncompetitive with respect to pyruvate for during the present investigation, and it pre- D-LDH at both pH values and at pH 5.4 for sented the problem of how this L(+)-lactate is L+LDH. produced before the D(-) isomer during the Effect of divalent metal ions and sulfhy- growth cycle. The total absence of any lactate dryl-binding reagents. When the effect of racemizing activity in crude extracts of P. six divalent metal ions was tested on D-LDH cerevisiae, together with reports of NAD- and L+LDH, only HgCl2 and CuSO, inhibited dependent D- and L-lactate dehydrogenases in pyruvate reduction, with HgCl2 generally caus- other pediococci (6, 14) clearly reveal that two on September 19, 2016 by University of Queensland Library ing the more potent inhibition (Table 4). Chlo- lactate dehydrogenases are the vehicles for rides of calcium, cobalt, magnesium, and racemic lactate production by this organism. manganese showed no effect, either positive Kinetic and physical properties of NAD- or negative, at a final concentration of 10 mM. dependent D- and L-lactate dehydrogenases Sulfhydryl-binding reagents, p-hydroxymercuri- from P. cerevisiae can be compared with those benzoate and iodoacetamide, were tested as from other organisms producing racemic lactic inhibitors of pyruvate reduction. Whereas acid, as studied by other workers. Michaelis iodoacetamide was without effect, p-hydroxy- constants for lactate and NAD, as well as pH mercuribenzoate inhibited both enzymes (Ta- optima for lactate oxidation for both enzymes, ble 4). are remarkably similar to those previously re- Molecular weights. The molecular weights ported for P. pentosaceus (6) and L. plantarum of P. cerevisiae D-LDH and L+LDH were (4). Comparison of the D-LDH can proceed estimated by gel filtration, by electrophoresis in further, with the enzyme from P. cerevisiae exhibiting heat lability and resistance to oxa- TABLE 4. Inhibition of D-LDH and L+LDH from P. mate inhibition similar to this enzyme from cerevisiae by compounds of regulatory significance and by divalent metal ions and TABLE 5. Estimated molecular weights of P. cerevisiae Concn causing 50% inhibition (mM) lactate dehydrogenases

Determinationa D-LDH . L+LDH Mol wt Method pH 5.4 pH 7.4 pH 5.4 pH 7.4 L+LDH D-LDH ATP ...... 0.8 0.6 1.2 NIb Gel filtration on Sephadex G-200 139,000 71,000 ADP ...... 3.0 2.7 1.0 4.5 Polyacrylamide gel electrophoresis GTP ...... 3.0 3.3 10 (44)c NI A. Nondenaturing gels by NAD ...... 10 (41)c 3.0 5.0 3.5 method of Hedrick and HgCl2 ...... 0.01 1.6 0.02 0.03 Smith (13) CuSO4 ...... 10 (34)c 0.5 0.2 1.5 i. Gel system of Davis (2) -a 72,000 p-Hydroxymer- ii. Modified gel system of 136,000 73,000 curibenzoate 0.26 0.12 0.6 0.6 (43)C Weber and Osborn (22)b a adeno- B. Denaturing gels by method 37,000 39,000 ATP, Adenosine 5-triphosphate; ADP, and gel system of Weber and sine 5-'-diphosphate; GTP, guanosine 5'-diphosphate. Osbom (22) b NI, Not inhibitory at a final concentration of 10 mM. aCould not be determined since L+LDH rapidly loses c Figure in parentheses indicates percent inhibition activity at alkaline pH. caused by the listed inhibitor concentration. b Gel system modified by omitting sodium dodecyl sulfate. 606 GORDON AND DOELLE J. BACTERIOL. assay were sufficient to protect the enzyme from Bovine serum A 8 the sulfhydryl inhibitor. albumin-dimer Molecular size and arrangement of both lactate dehydrogenases from P. cerevisiae are of 7 interest since they conform to the general pat- tern that is gradually becoming apparent for all lactic acid bacteria. The dimeric arrangement Downloaded from CL - Hexokinase for D-LDH from P. cerevisiae with a molecular 0 6[ weight of about 70,000 shows a close similarity to the enzymes from Leuconostoc lactis (11, 16) 51 and Lactobacillus leichmannii (11), whereas the 4-I B a tetrameric arrangement for L-+ LDH from P. cerevisiae with a molecular weight of about z Bovine serum 4 albumin-monomer 140,000 shows a close similarity to the enzyme

from Streptococcus cremoris (7). Native molec- http://jb.asm.org/ ular weights of both of these enzymes from P. 3 Ovalbumin cerevisiae are comparable to values reported for I. many other lactic acid bacteria (5, 8, 10). 40 60 80 100 120 140 Metabolic control of D-LDH and L+LDH Molecular Weight (x 103) from P. cerevisiae appears to be similar to the mechanisms described previously for L. lactis FIG. 5. Standard curve for the estimation of the (11), with pyruvate reduction by both enzymes native molecular weights of P. cerevisiae D-LDH and NAD and L+LDH by electrophoresis in nondenaturing poly- being subject to product inhibition by on September 19, 2016 by University of Queensland Library acrylamide gels (pH 7.0) by the method of Hedrick to energy-dependent inhibition by adenosine and Smith (13). A, L+LDH; B, D-LDH. Standard triphosphate. Although these inhibitory effects protein molecular weights: ovalbumin, 45,000; bovine are not as profoundly affected by pH as they are serum albumin, 67,000; dimer, 134,000; hexokinase, in leuconostocs, it seems that they still form the 99,000. basis for the regulation of total lactate production by P. cerevisiae. However, this regulatory other lactic acid bacteria (4, 8, 11, 16, 19). phenomenon fails to explain the reason for the However, in regard to these characters, the sequential production of the two isomers of L+LDH from P. cerevisiae appears to resemble lactic acid in cultures of P. cerevisiae. It is our other D-LDH in that it is heat labile and belief that culture pH is the determining factor oxamate does not inhibit lactate oxidation. This in this production. is in contradiction to the behavior of L+LDH In the present study it has been found that from various lactobacilli (4, 10), and only fur- D-LDH and L+LDH from P. cerevisiae react ther investigation will show whether these char- differently to change in pH depending upon the acters are common to most lactic acid bacteria pyruvate concentration in the assay (Fig. 3). or if they are confined solely to the lactobacilli. Although Mizushima and Kitahara (18) re- The effect of pyruvate analogues on pyruvate ported that the intracellular pyruvate concen- reduction by both enzymes is worthy of men- tration in L. plantarum is approximately 5.2 tion. a-Ketobutyrate can be utilized as a redu- mM, it would appear that the actual concentra- cible substrate by both D-LDH and L+LDH tion in P. cerevisiae is much lower than this. from P. cerevisiae, which is also the case in L. Since the specific activity of pyruvate reduction plantarum (4). However, oxamate is a potent in a crude extract of P. cerevisiae is 8.7 U/mg of inhibitor of pyruvate reduction by L. plantarum protein while pyruvate kinase (EC 2.7.1.40) is L+LDH with 50% inhibition resulting at 0.15 extremely difficult to detect (G. Gordon and H. mM oxamate, whereas D-LDH is unaffected. Doelle, unpublished observations), it seems un- Under similar circumstances neither D-LDH likely that pyruvate would be able to accumu- nor L+LDH from P. cerevisiae is inhibited. late in these cells and thus reach an intracellu- The slight inhibition of L+ LDH from P. lar concentration as high as 5 mM. Therefore, cerevisiae caused by p-hydroxymercuribenzoate the pH profile most likely to correspond to is unusual since the presence of essential sulfhy- physiological conditions is that obtained using dryl groups in the enzyme protein is indicated low pyruvate concentrations (0.5 mM). by its absolute requirement for reducing com- During the growth cycle of P. cerevisiae, pounds during purification. Perhaps the DTT L( +)-lactate production commences with initia- added to the partially purified preparation or tion of growth when the pH of the medium is the relatively high substrate levels used for about 6.7 (Fig. 1), whereas this production VOL. 121, 1975 LACTATE DEHYDROGENASES FROM P. CEREVISIAE 607

ceases at almost the same time as D(-)-lactate 8. Garland, R. C. 1973. Purification and properties of D( )-lactate dehydrogenase from Leuconostoc mesent- production commences (when the pH is about eroides. Arch. Biochem. Biophys. 157:36-43. 5.0). Actively fermenting L. plantarum cells 9. Garvie, E. I. 1967. The production of L(+) and D(-) lactic have an intemal pH of 5.4 (19) and, in the acid in cultures of some lactic acid bacteria, with a absence of any contrary evidence, it seems special study of Lactobacillus acidophilus NCDO2. J. Dairy Res. 34:31-38. reasonable to assume that the intracellular pH 10. Gasser, F., M. Doudoroff, and R. Contopoulos. 1970. Downloaded from is similar to that of the medium. By reference to Purification and properties of NAD-dependent lactic Fig. 3A, it can be seen that, as the pH falls from dehydrogenases of different species of Lactobacillus. J. about 6.5 to 5.0, L+LDH attains maximum Gen. Microbiol. 62:241-250. 11. Gordon, G. L., and H. W. Doelle. 1974. Molecular aspects activity while D -LDH activity is still relatively for the metabolic regulation of the nicotinamide ade- low. At this pH in the culture, D(-)-lactate nine dinucleotide-dependent D( )-lactate dehydrogen- production commences with the pH falling to ase from Leuconostoc. Microbios 9:199-215. about 4.0 as a result, which can be explained by 12. Gornall, A., C. Bardowell, and M. David. 1949. Deter- mination of serum proteins by means of the biuret the activity maximum of D-LDH at a pH of 3.6. reagent. J. Biol. Chem. 177:751-756. However, this apparently carefully regulated 13. Hedrick, J. L., and A. J. Smith. 1968. Size and charge http://jb.asm.org/ total production of one isomer of lactic acid isomer separation and estimation of molecular weights followed by a switch to the total production of of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164. the other does not appear to be common among 14. Hiyama, T., S. Fukui, and K. Kitahara. 1968. Purifica- all racemic lactate-producing Lactobacillaceae. tion and properties of lactate racemase from Lac- For instance, the percentage of L(+) acid in the tobacillus sake. J. Biochem. (Tokyo) 64:99-107. culture changes little during growth of L. 15. Hohorst, H.-J. 1963. L(+)-Lactate determination with lactic dehydrogenase and DPN, p. 266-270. In H. U. plantarum (9), which is reflected in the fact Bergmeyer (ed.), Methods of enzymatic analysis. Aca- that both lactate dehydrogenases from this demic Press Inc., New York. on September 19, 2016 by University of Queensland Library organism display an optimum for pyruvate 16. Hontebeyrie, M., and F. Gasser. 1973. Separation et reduction at pH 6.0 (17). purification de la D-lactico-deshydrogenase et de la glucose-6-phosphate deshydrogenase de Leuconostoc lactis. Etude de quelques propietes. Biochimie 55: ACKNOWLEDGMENTS 1047-1056. This work was carried out with finances from the Australian 17. Mizushima, S., T. Hiyama, and K. Kitahara. 1964. Universities Commission and with support from a Common- Quantitative studies on glycolytic enzymes in Lactoba- wealth Postgraduate Research Award (G.L.G.). cillus plantarum. III. Intracellular activities of reverse reaction of D- and L-lactate dehydrogenases during glucose fermentation. J. Gen. Appl. Microbiol. (Tokyo) LITERATURE CITED 10:33-44. 1. Breed, R. S., E. D. G. Murray, and N. R. S. Smith (ed.). 18. Mizushima, S., and K. Kitahara. 1964. Quantitative 1957. Bergey's manual of determinative bacteriology, studies on glycolytic enzymes in Lactobacillus 7th ed. The Williams & Wilkins Co., Baltimore. plantarum. II. Intracellular concentrations of glycolytic 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and intermediates in glucose-metabolizing washed cells. J. application to serum proteins. Ann. N.Y. Acad. Sci. Bacteriol. 87:1429-1435. 121:404-427. 19. Mizushima, S., Y. Machida, and K. Kitahara. 1963. 3. DeMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A Quantitative studies on glycolytic enzymes in Lac- medium for the cultivation of lactobacilli. J. Appl. tobacillus plantarum. I. Concentration of inorganic Bacteriol. 23:130-135. ions and coenzymes in fermenting cells. J. Bacteriol. 4. Dennis, D., and N. 0. Kaplan. 1960. D- and L-lactic acid 86:1295-1300. dehydrogenases in Lactobacillus plantarum. J. Biol. 20. Stetter, K. O., and 0. Kandler. 1973. Untersuchungen Chem. 235:810-818. zur Entstehung von DL-Milchsiiure bei Lactobacillen 5. Dennis, D., M. Reichlin, and N. 0. Kaplan. 1965. Lactic und Characterisierung einer Milchsiiureracemase bei acid racemization. Ann. N.Y. Acad. Sci. 119:868-876. einigen Arten der Untergattung Streptobacterium. 6. Doelle, H. W. 1971. Nicotinamide adenine dinucleotide- Arch. Mikrobiol. 94:221-247. dependent and nicotinamide adenine dinucleotide- 21. Warburg, O., and W. Christian. 1942. Isolierung und independent lactate dehydrogenases in homofermenta- Kristallisation des Garungsferments Enolase. Bio- tive and heterofermentative lactic acid bacteria. J. chem. Z. 310:384-421. Bacteriol. 108:1284-1289. 22. Weber, K., and M. Osborn. 1969. The reliability of 7. Dynon, M. K., G. R. Jago, and B. E. Davidson. 1972. The molecular weight determinations by dodecylsulphate- subunit structure of lactate dehydrogenase from Strep- polyacrylamide gel electrophoresis. J. Biol. Chem. tococcus cremoris US3. Eur. J. Biochem. 30:348-353. 244:4406-4412.