Hostname: page-component-5db6c4db9b-k8dct Total loading time: 0 Render date: 2023-03-24T00:01:16.894Z Has data issue: true Feature Flags: { "useRatesEcommerce": false } hasContentIssue true

Intracellular pH and the role of D-lactate dehydrogenase in the production of metabolic end products by Leuconostoc lactis

Published online by Cambridge University Press:  01 June 2009

Richard J. Fitzgerald
National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Irish Republic Department of Biochemistry, University College, Cork, Irish Republic
Shawn Doonan
Department of Biochemistry, University College, Cork, Irish Republic
Larry L. McKay
Department of Food Science and Nutrition, University of Minnesota, St Paul, MN 55108, USA
Timothy M. Cogan
National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Irish Republic


The kinetics of lactate dehydrogenase from Leuconostoc lactis NCW1 were studied. The pH optimum for the enzyme depended on the concentration of pyruvate used in the assay and the enzyme displayed an ordered mechanism with respect to substrate binding. The Km for pyruvate and NADH and the Vmax of the enzyme decreased 20–, 30– and 6-fold respectively as the pH decreased from 8·0 to 5·0. No activators were found and none of the intermediates of the phosphoketolase pathway tested inhibited the enzyme. ATP, ADP, GTP and NAD+ were inhibitory. The intracellular volume (Volin) and intracellular pH (pHin) decreased as the extracellular pH (pHex) decreased. Co-metabolism of citrate and glucose affected the Volin but did not affect the pHin, which decreased by 0·6 units per unit change in pHex; at pH 7·0, the pHin and pHex were equal. The results suggest that pHin may play a role in determining the production of diacetyl and acetoin at low pH by Leuconostoc.

Original Articles
Copyright © Proprietors of Journal of Dairy Research 1992

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)



Baronofsky, J. J., Schreurs, W. J. A. & Kashket, E. R. 1984 Uncoupling by acetic acid limits growth of and acetogenesis by Clostridium thermoaceticum. Applied and Environmental Microbiology 48 11341139Google ScholarPubMed
Booth, I. R. 1985 Regulation of cytoplasmic pH in bacteria. Microbiologial Reviews 49 359378Google ScholarPubMed
Cleland, W. W. 1967 The statistical analysis of enzyme kinetic data. Advances in Enzymology 29 132Google ScholarPubMed
Cleland, W. W. 1970 Steady state kinetics. In The Enzymes, 3rd edn, vol. 2, pp. 165 (Ed. Boyer, P. D.). New York: Academic PressGoogle Scholar
Cogan, T. M. 1987 Co-metabolism of citrate and glucose by Leuconostoc spp.: effects on growth, substrates and products. Journal of Applied Bacteriology 63 551558CrossRefGoogle Scholar
Cogan, T. M., O'Dowd, M. & Mellerick, D. 1981 Effects of pH and sugar on acetoin production from citrate by Leuconostoc lactis. Applied and Environmental Microbiology 41 18Google ScholarPubMed
De Man, J. C., Rogosa, M. & Sharpe, M. E. 1960 A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23 130135CrossRefGoogle Scholar
De Moss, R. D., Bard, R. C. & Gunsalus, I. C. 1951 The mechanism of the heterolactic fermentation: a new route of ethanol formation. Journal of Bacteriology 62 499511Google Scholar
Dixon, M. & Webb, E. C. 1979 In Enzymes, 3rd edn, pp. 358360 (Eds Dixon, M., Webb, E. C., Thorne, C. J. R. and Tipton, K. F.). London: Longman Group LimitedGoogle Scholar
Drinan, D. F., Tobin, S. & Cogan, T. M. 1976 Citric acid metabolism in hetero- and homo-fermentative lactic acid bacteria. Applied and Environmental Microbiology 31 481486Google Scholar
Fawcett, C. P., Ciotti, M. M. & Kaplan, N. O. 1961 Inhibition of dehydrogenase reactions by a substance formed from reduced diphosphopyridine nucleotide. Biochimica et Biophysica Acta 54 210212CrossRefGoogle ScholarPubMed
FitzGerald, R. J., Doonan, S. & Cogan, T. M. 1986 Purification and characterization of the D-lactate dehydrogenase from Leuconostoc lactis. International Journal of Biochemistry 18 3138CrossRefGoogle Scholar
Fordyce, A. M., Crow, V. L. & Thomas, T. D. 1984 Regulation of product formation during glucose or lactose limitation in non-growing cells of Streptococcus lactis. Applied and Environmental Microbiology 48 332337Google ScholarPubMed
Garvie, E. I. 1980 Bacterial lactate dehydrogenases. Microbiological Reviews 44 106139Google ScholarPubMed
Gordon, G. L. & Doelle, H. W. 1974 Molecular aspects for the metabolic regulation of the nicotinamide adenine dinucleotide-dependent D( −)-lactate dehydrogenase from Leuconostoc. Microbios 9 199215Google ScholarPubMed
Gordon, G. L. & Doelle, H. W. 1975 Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases. Journal of Bacteriology 121 600607Google ScholarPubMed
Gunsalus, I. C. & Gibbs, M. 1952 The heterolactic fermentation. 2. Position of C14 in the products of glucose dissimilation by Leuconostoc mesenteroides. Journal of Biological Chemistry 194 871875Google ScholarPubMed
Kashket, E. R., Blanchard, A. G. & Metzger, W. C. 1980 Proton motive force during growth of Streptococcus lactis cells. Journal of Bacteriology 143 128134Google ScholarPubMed
Lineweaver, H. & Burk, D. 1934 The determination of enzyme dissociation constants. Journal of the American Chemical Society 56 658666CrossRefGoogle Scholar
Lowry, O. H. & Passonneau, J. V. 1972 A Flexible System of Enzymatic Analysis pp. 320. New York: Academic PressCrossRefGoogle Scholar
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 1951 Protein estimation with the Folin phenol reagent. Journal of Biological Chemistry 193 265275Google Scholar
McDonald, L. C., Fleming, H. P. & Hassan, H. M. 1990 Acid tolerance of Leuconostoc mesenteroides and Laclobacillus plantarum. Applied and Environmental Microbiology 56 21202124Google ScholarPubMed
Maloney, P. C. 1983 Relationship between phosphorylation potential and electrochemical H+ gradient during glycolysis in Streptococcus lactis. Journal of Bacteriology 153 14611470Google ScholarPubMed
Marier, J. R. & Boulet, M. 1958 Direct determination of citric acid in milk with an improved pyridine-acetic anhydride method. Journal of Dairy Science 41 16831692CrossRefGoogle Scholar
Nannen, N. L. & Hutkins, R. W. 1991 Intracellular pH effects in lactic acid bacteria. Journal of Dairy Science 74 741746CrossRefGoogle Scholar
Perrin, D. D. & Dempsey, B. 1974 Buffers for pH and Metal Ion Control. London: Chapman and Hall Laboratory ManualsGoogle Scholar
Poolman, B., Driessen, A. J. M. & Konings, W. N. 1987 a Regulation of solute transport in streptococci by external and internal pH values. Microbiological Reviews 51 498508Google ScholarPubMed
Poolman, B., Nijssen, R. M. J. & Konings, W. N. 1987 b Dependence of Streptococcus lactis phosphate transport on internal phosphate concentration and internal pH. Journal of Bacteriology 169 53735378CrossRefGoogle ScholarPubMed
Snoswell, A. M. 1963 Oxidized nicotinamide-adenine dinucleotide-independent lactate dehydrogenases of Lactobacillus arabinosus 17.5. Biochimica et Biophysica Acta 77 719CrossRefGoogle ScholarPubMed
Speckman, R. A. & Collins, E. B. 1968 Diacetyl biosynthesis in Streptococcus diacetilactis and Leuconostoc citrovorum. Journal of Bacteriology 95 174180Google ScholarPubMed
Thomas, T. D. 1976 Regulation of lactose fermentation in group N streptococci. Applied and Environmental Microbiology 32 474478Google ScholarPubMed
Thomas, T. D., Ellwood, D. C. & Longyear, V. M. C. 1979 Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. Journal of Bacteriology 138 109117Google ScholarPubMed
Wilkinson, G. N. 1961 Statistical estimations in enzyme kinetics. Biochemical Journal 80 324332CrossRefGoogle ScholarPubMed