Purification and Properties of Nicotinamide Adenine Dinucleotide-Dependent D- and L- Lactate Dehydrogenases in a Group N Streptococcus

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1972, P Copyright American Society for Microbiology Vol. 111, No. 2 Printed in U.S.A. Purification and Properties of Nicotinamide Adenine Dinucleotide-Dependent D- and L- Lactate Dehydrogenases in a Group N Streptococcus L. MOU, D. P. MULVENA, H. A. JONAS, AND G. R. JAGO Russell Grimwade School of Biochemistry, University of Melboumne, Parkville, Victoria 3052, Australia, and C.S.I.R.O., Division of Food Research, Dairy Research Laboratory, Highett, Victoria 3190, Australia Received for publication 13 March 1972 Streptococcus lactis strain 760, a group N streptococcus, was found to possess nicotinamide adenine dinucleotide-dependent dehydrogenase activities for both the L(+) and the D(-) isomers of lactic acid. The two enzymes were isolated and purified and were found to differ with respect to ph optima, activation by fructose-1,6-diphosphate, ph and heat stability, and the temperature at which each enzyme was formed in the organism during growth. The presence of a racemase for lactic acid was not detected by the methods used. It has been reported that bacterial species belonging to the genus Streptococcus produce only the L(+) isomer of lactic acid during the fermentation of glucose (1, 8). In a recent survey of lactate dehydrogenase activities in 32 strains of group N streptococci, selected for their ability to produce lactic acid rapidly in milk culture, it was found that all but one strain possessed only a single nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (LDH; EC ), and this was specific for L(+)-lactic acid (H. A. Jonas, M.S. thesis, Univ. of Melbourne, 1968). The exception was strain 760 which possessed NAD-dependent LDH activity which was specific for both the L(+) and the D(-) isomers of lactic acid. The present investigation was made to determine whether the L- and D-LDH activity observed in this strain was due to the presence of two stereospecific LDH species or to the presence of a racemase capable of converting the D(-) to the L(+) isomer of lactic acid. MATERIALS AND METHODS Strain 760 was obtained from United Dairies Research Laboratories, Wood Lane, London, in Identification of the strain was confirmed as a group N streptococcus of the species Streptococcus lactis by the standard biochemical and serological reactions (5). The organism was maintained by daily subculture in autoclaved (115 C for 10 min) skim milk (10% solution of milk solids, w/v) and grown in quantity in broth containing per liter of tap water: 30 g of tryptone (Difco), 10 g of yeast extract (Difco), 30 g of lactose, 5 g of K2HPO4, and 2 g of beef extract (Oxoid). The medium was filtered (Whatman no. 1 paper) and autoclaved at 121 C for 20 min. Each 5-liter batch of broth was inoculated with 10 ml of a coagulated skim milk culture and incubated at 30 C for 16 hr. The ph of the growth medium was maintained at 6.3 by the addition of 10 M NaOH controlled by a magnetic valve connected to a Radiometer Titrator, model TTTA3. The cells were collected by centrifugation at 24,000 x g for 20 min at 4 C, washed with 0.9% NaCl, and resuspended in 0.1 M sodium phosphate buffer, ph 7.0 (1 g wet weight/4 ml of buffer). Cell-free extracts were prepared by extruding suspensions of washed cells through a French pressure cell (3, 9) at a constant pressure of 10 tons/in2 (1,575 kg/cm2). Unbroken cells and cellular debris were removed by centrifugation at 35,000 x g for 20 min at 4 C. The supernatant fluid was dialyzed overnight against 0.01 M sodium phosphate, ph 7.0, at 4 C. The protein and nucleic acid concentration of the extract was estimated from the absorbancy at 280 and 260 nm according to the nomograph devised by Adams (nomograph supplied by the California Corporation for Biochemical Research, 2625 Madford St., Los Angeles 36, Calif.). Protein was also estimated by the method of Lowry et al. (4). NAD-dependent LDH activity was measured either by pyruvate reduction or by lactate oxidation. Details of the reaction mixtures used are given in the legends to figures and tables. Initial reaction rates were estimated from the rate of decrease (or increase) in the absorbancy of NADH at 340 nm, measured in 1-cm cells on a Zeiss spectrophotom- 392

2 VOL. 111, 1972 LACTATE DEHYDROGENASES FROM S. LACTIS eter, model PMQII, coupled to a Rikadenki recorder, model B-14, in the period immediately following the addition of enzyme extract. One unit of LDH activity was defined as that amount of enzyme causing a change in the absorbancy at 340 nm of 0.1/min. This corresponds to the oxidation or reduction of Mmole of coenzyme/min. Specific activity was defined as LDH units per milligram of protein. RESULTS Formation of L(+)- and D(+)-lactic acids. Both L(+)- and D(-)-lactic acid were produced in the growth medium by strain 760. The organism was grown in broth containing 5% lactose at a controlled ph of 6.3 until a considerable amount of lactic acid had been formed. After centrifugation, the supernatant fluid was acidified to ph 1 with concentrated H2SO and continuously extracted with ether for 72 hr. After extraction, the ether was removed by evaporation and the residue of organic acids was dissolved in 5 ml of water and neutralized with concentrated NaOH. As a control, the growth medium, without organisms, was subjected to the same treatment. This neutralized extract of organic acids was used as a source of lactate in assay mixtures containing NAD+ and cell-free extracts of Streptococcus cremoris strain US3 [source of LDH specific for the L(+) isomer] or cell-free extracts of Lactobacillus bulgaricus strain LB1 [source of LDH specific for the D(-) isomer]. Reduction of NAD+ occurred in both assay mixtures, indicating that strain 760 had produced both D(-) and L(-) lactic acids. From the initial rate of reduction of NAD+ in each assay, the ratio of the L( -) isomer to that of the D(-) isomer was estimated to be 36: 1. Cell-free extracts of strain 760 were examined for racemase activity by incubating the extracts at 30 C with D(-)-lactic acid and testing for the formation of L(+)-lactic acid. The incubation mixture, contained in a total volume of 3.0 ml: 2.1 ml of 0.1 M triethanolamine-hydrochloride buffer, ph 7.9; 0.8 ml of 0.75 M D(-)-lactic acid, and 0.1 ml (2.8 mg of protein) of cell-free extract. The controls contained either no substrate or no enzyme. Sam- 393 ples (1.0 ml) were taken at 0, 30, and 60 min, heated at 100 C for 5 min, and assayed for L(+)-lactic acid by using an L(+)-specific LDH prepared from S. cremoris US3. No L(+)-lactic acid was detected in the incubation mixture by this method, and it was concluded that racemase activity was absent in strain 760. Separation and purification of the L(+)- and D(- )-LDH species. Fractionation of cellfree extracts of strain 760 was carried out sequentially by the addition of streptomycin sulfate (to remove the nucleic acid component), by the addition of ammonium sulfate, by chromatography on diethylaminoethyl (DEAE)- Sephadex and DEAE-cellulose, and by gel filtration on Sephadex G-100. All enzyme activities were determined in the absence of fructose-1, 6-diphosphate (FDP) using NAD+, L-, or D-lactate as substrates. Step 1: A 10% solution (w/v) of streptomycin sulfate was added dropwise to 176 ml of the dialyzed cell-free extract with constant stirring at 4 C until no further precipitation of the nucleic acid component was obtained. The precipitate was removed by centrifugation at 35,000 x g for 20 min and discarded. The supernatant fluid was dialyzed overnight against 0.01 M sodium phosphate buffer, ph 7.0, at 4 C. Analysis of the cell-free extract before and after streptomycin sulfate treatment showed that 90% of the nucleic acid component had been precipitated but most of the LDH activity and protein had been retained. Step 2: Stepwise fractionation with ammonium sulfate at ph 7.0 of the streptomycin sulfate-treated extract indicated that the bulk of the LDH activity was precipitated in the 50 to 60% fraction. However, because the 40 to 50% fraction also contained a significant amount of LDH activity, the combined 40 to 60% fraction was dissolved in a buffer containing 0.05 M sodium phosphate and 0.1 M NaCl, ph 7.0, and retained for further purification. Step 3: A portion (866 mg of protein) of the 40 to 60% ammonium sulfate fraction, which had been dialyzed against 0.05 M sodium phosphate buffer (ph 7.0) containing 0.1 M NaCl, was applied to a DEAE-Sephadex A-50 column (78 by 1.5 cm) equilibrated with the same buffer at 4 C. The L- and D-LDH species were eluted from the column by using a linear sodium chloride gradient (0.1 to 0.5 M) in 0.05 M sodium phosphate buffer, ph 7.0. Fractions of average volume 10 ml were collected and assayed. As shown in Fig. la the two enzymes were not separated by this method. However, there was a significant increase in the specific activity of both enzymes. Step 4: Fraction A (see Fig. la) from the DEAE-Sephadex column was applied to a DEAE-cellulose (Whatman DEII) column (120 by 1.5 cm) equilibrated with 0.05 M sodium phosphate buffer, ph 7.0 at 4 C. The dehydrogenase activities were eluted from the column by using a linear sodium chloride gradient (0 to 0.5 M) in 0.05 M sodium phosphate buffer, ph 7.0. Fractions of average volume 10 ml were collected and assayed. As shown in Fig.

3 394 MOU ET AL. J. BACTERIOL. lb, the major peaks of L- and -LDH activity were separated but not sufficiently to give samples of one enzyme which was free of the other. Fractions were pooled to give two fractions, A and B (see Fig. lb) in which the L- LDH and the D-LDH predominated, respectively. Step 5: Fractions A and B from the DEAEcellulose column were applied separately to two Sephadex G-100 columns (130 by 1.5 cm) equilibrated with 0.05 M sodium phosphate buffer, ph 7.0 at 4 C. The L- and D-LDH species were eluted from the columns by using 0.05 M sodium phosphate buffer, ph 7.0. As shown in Fig. lc and d, samples of both the D- and the L-enzymes could be obtained, free of contaminating activities, by this procedure. A summary of the purification steps with measures of their relative efficiency is presented in Table 1. -(a) FRACTION A 60~ ~ ~ ~ ~ ~ < 300 (c) 6115 z i S J 20 (b) FRACTION FRACTION B L!eDL0~~=410 ~~ ~ ~ ~ ~ * ELUTION VOLUME (ml) 2-(d) 04 oc.-, 00 FIG. 1. Elution profiles of L- and D-LDH activity: (a) DEAE-Sephadex column; (b) DEAE-cellulose column; (c) Sephadex G-100 column (fraction B); and (d) Sephadex G-100 column (fraction A). 0, D-LDH; A, L-LDH; *, protein. Assay of L- and D-LDH activity was carried out in the reaction mixture described in Table 2, and protein was assayed as described in Materials and Methods. TABLE 1. Summary of the steps involved in the purification of L- and D-lactate dehydrogerise (LDH) from cell-free extracts of strain 760Y Total activity' Total Specific activity (enzyme units) Total nucleic (units/mg of % Yield D-LDH/ Step Fraction protein acid protein) L-LDH (mg) acid D-LDH L-LDH (mg) D-LDH L-LDH D-LDH L-LDH Cell-free extract 6,160 3,696 4,840 1, Streptomycin sulfate 5,975 3,585 3, supernatant 2 (NH4),S04 fraction ( , %) 3 DEAE-Sephadex fraction A 2, DEAE-cellulose fraction A fraction B 1, Sephadex G-100 fraction mlc (Fig. ld) fraction mld (Fig. lc) I_I_I_I a Each step has been described in detail in the text. b D- and L-LDH activity was assayed in the reaction mixture detailed in Table 2. C This fraction is the purified L(+)-LDH used in this study. d This fraction is the purified D(-)-LDH used in this study E o r-

4 VOL. 111, 1972 LACTATE DEHYDROGENASES FROM S. LACTIS 395 Distinguishing properties of the L(+)- and D(-)-LDH species. The effect of ph on the rate of reduction of pyruvate by the L- and D- LDH species is shown in Fig. 2. In the absence of FDP, which has been shown to activate streptococcal LDH species (7), the ph optimum of the L-LDH was 8.0. This activity in the absence of FDP was in contrast to the S. faecalis L-LDH which has been shown by Wittenberger and Angelo (6) to have an absolute requirement for FDP activation. In the presence of excess FDP (10 mm), the ph optimum of the L-LDH changed to a broad plateau between ph 7.0 and ph 5.0. It should be noted that within this ph range, outside of which the organism does not grow, the L-LDH enzyme showed an absolute requirement for FDP activation. The ph optimum for the D-LDH, which was not activated by FDP, was approxi-._ U-, 0 -J CITRATE-PHOSPHATE BUFFER PHOSPHATE BUFFER mately 8.5 in both the presence and absence of FDP. In a preliminary investigation, it was observed that the L-LDH rapidly lost activity at ph values above 7.5 and below 5.0, at 20 C, whereas the D-LDH was essentially stable over the ph range 4.5 to 9.5. The rate of inactivation of the L-LDH at ph 8.0 was increased when the temperature was raised to 40 C. However, in the presence of FDP the L-LDH was quite stable at the above ph and temperature (Fig. 3). Only at 40 C did the D-LDH show gradual inactivation at ph 8.0, and this inactivation was not prevented by the addition of FDP (Fig. 3). Extracts incubated at ph 7.0 and 40 C, in the presence of FDP, showed no change in either L- or D-LDH activity. Separate batches of strain 760 were grown in broth at 25, 30, 35, and 40 C for 16 hr at a constant ph of 6.3. The cell-free extracts, prepared from each batch of cells, were assayed for L- and D-LDH activity. As shown in Table 2, an increase in the temperature of growth from 30 to 40 C decreased markedly the levels of D-LDH present in the cells in contrast to the levels of L-LDH which remained fairly constant. The specific activity of both enzymes was highest in cells grown at 25 C. However, the activity of the L-LDH, which can be activated by FDP, is potentially higher than that shown in Table 2 where activities were compared in the absence of FDP. DISCUSSION Although it has been reported (1, 8) that streptococci form only L(+)-lactic acid from lactose, the results of this investigation show 4 CO a 120 r A L-LDH (+FDP) D-LD (FDP) ph FIG. 2. ph optima for L-LDH (A) and D-LDH (0) in the presence and absence of 10 mm FDP. The reaction mixtures contained in a total volume 3.0 ml: 2 ml of 0.1 M sodium phosphate or phosphatecitrate (0.1 M with respect to phosphate) buffer at the ph values indicated; 0.1 ml of 4 mm NADH; 0.24 ml of 1 M sodium pyruvate; 0.1 ml of 300 mm FDP (where used); and 0.1 ml of purified enzyme (15,ug of protein for D-LDH and 36 ;tg of protein for L- LDH). L-LDH (-FDP) 0 _0 Z TIME OF INCUBATION (min.) FIG. 3. Effect of FDP on the denaturation of L- (A) and D- (0) LDH at ph 8.0 and 40 C. Partially purified cell-free extract (40-60% ammonium sulfate fraction) was adjusted to ph 8.0 and incubated at 40 C with and without 10 mm FDP. At the specified time intervals, 0.1-ml samples (containing 1.3 mg of protein) of the incubation mixtures were assayed in the reaction mixture described in Table 2. Results are expressed as per cent LDH activity of unheated enzyme.

5 396 MOU ET AL. J. BACTERIOL. TABLE 2. Effect of temperature of growth on the formation of L- and D-lactate dehydrogenase (LDH)a Specific activity Growth (units/mg of protein) D-LDH/L-LDH temp (C) D-LDH L-LDH a Strain 760 was grown for 16 hr in broth at a constant ph of 6.3 and at the temperatures indicated. L- and D-LDH activity was assayed in the cell-free extracts prepared from each batch of cells by using a reaction mixture which contained in a total volume 3 ml: 2.2 ml of 0.1 M triethanolamine-hydrochloride, ph 7.9; 0.5 ml of 0.5 M sodium L- or D-lactate; 0.2 ml of 0.01 M NAD+; and 0.1 ml (1 mg of protein) of cellfree extract. that strain 760 produces both the L(+) and the D( -) isomers. The absence of racemase activity and the presence of the two distinct NAD-dependent LDH species, each specific for either the L(+) or the D(-) isomers of lactic acid, in cell-free extracts of strain 760 indicate that the two isomers are formed by different enzymes. The two enzymes differed with respect to ph optima, activation by FDP, ph and heat stability, and the temperature at which each enzyme appeared in the organism during growth. The failure of the D-LDH to appear in cells grown at 40 C may be related to the heat lability of this enzyme which is not protected by FDP. Although the levels of D-LDH always exceeded that of L-LDH in strain 760 when grown at its optimal growth temperature (30 C; Tables 1 and 2) it was found in the growth medium, however, that approximately 36 times as much L(+)-lactic acid as D(-)-lactic acid had been produced. This quantitative difference in the formation of the two isomers could result from the activation of the L-LDH by metabolically produced FDP. Since these organisms satisfy their energy requirements by fermenting glucose via the glycolytic pathway, it would appear that the FDP formed during glycolysis could serve as a form of metabolic control over the synthesis of L(+) lactate. The relative amounts of L(+)- and D(-)-lactic acids formed in the presence of carbohydrates, other than lactose, in the growth medium, was not investigated. By contrast, the combined action of the NAD-dependent D- and L-LDH species in Lactobacillus plantarum which are not activated by FDP, has been shown to form a racemic mixture of the two isomers of lactate (2). ACKNOWLEDGMENTS This work was supported by grants from the Australian Dairy Industry Research Fund administered by the Australian Dairy Produce Board. The receipt of a University of Melbourne Research Scholarship (H.A.J.) is acknowledged. LITERATURE CITED 1. Bergey, D. H Manual of determinative bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore. 2. Dennis, D., and N. 0. Kaplan D- and L-lactic acid dehydrogenases in Lactobacillus plantarum. J. Biol. Chem. 235: French, C. S., and H. W. Milner Disintegration of bacteria and small particles by high-pressure extrusion, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York. 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Swartling, P. F Biochemical and serological properties of some citric acid-fermenting Streptococci from milk and dairy products. J. Dairy Res. 18: Wittenberger, C. L., and N. Angelo Purification and properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Streptococcus faecalis. J. Bacteriol. 101: Wolin, M. J Fructose-1, 6-diphosphate requirement of streptococcal lactate dehydrogenases. Science 146: Wood, W. A Fermentation of carbohydrate and related compounds, p. 59. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. 2. Academic Press Inc., New York. 9. Venderheiden, G. J., A. C. Fairchild, and G. R. Jago Construction of a laboratory press for use with the French pressure cell. Appl. Microbiol. 19: