European Food Research and Technology

, Volume 244, Issue 4, pp 603–609 | Cite as

Identification and characterization of glutamate dehydrogenase activity in wild Lactococcus lactis isolated from raw milk cheeses

  • Luz P. Gómez de Cadiñanos
  • Carmen Peláez
  • M. Carmen Martínez-Cuesta
  • Tomás García-Cayuela
  • Teresa Requena
Original Paper


The glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate, which initiates amino acid transamination during cheese ripening. This work has investigated the GDH activity in 39 wild isolates of Lactococcus lactis from raw milk cheeses. Only 25% of the isolates were GDH positive with NAD+ as the preferred cofactor. L. lactis IFPL953 showed the highest NAD-GDH activity. The GDH activity at the genetic level in the lactococcal isolates was analysed by PCR amplification of the gdh gene in genomic and plasmid DNA. The gdh gene arrangement of L. lactis IFPL953 in its plasmid location was similar to that in the reference strain GDH+ L. lactis TiL504, suggesting that both lactococci could harbour the same plasmid pGdh442 containing the gdh gene. L. lactis IFPL953 has previously demonstrated a remarkable α-ketoisovalerate decarboxylase activity, which along with its high GDH activity makes the strain particularly useful in enhancing cheese flavour formation.


Lactococcus lactis Glutamate dehydrogenase Amino acid catabolism Cheese aroma 



The authors acknowledge the funding from the Spanish MINEICO (AGL2012-35814, AGL2016-75951-R and P916PTE0233).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.


  1. 1.
    Smid EJ, Kleerebezem M (2014) Production of aroma compounds in lactic fermentations. Annu Rev Food Sci Technol 5:323–326CrossRefGoogle Scholar
  2. 2.
    Steele J, Broadbent J, Kok J (2013) Perspectives on the contribution of lactic acid bacteria to cheese flavour development. Curr Opin Biotechnol 24:135–141CrossRefGoogle Scholar
  3. 3.
    De la Plaza M, Fernández de Palencia P, Peláez C, Requena T (2004) Biochemical and molecular characterization of α-ketoisovalerate decarboxylase an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. FEMS Microbiol Lett 238:367–374CrossRefGoogle Scholar
  4. 4.
    De la Plaza M, Peláez C, Requena T (2008) Regulation of α-ketoisovalerate decarboxylase expression in Lactococcus lactis IFPL730. J Mol Microbiol Biotechnol 17:96–100CrossRefGoogle Scholar
  5. 5.
    Yvon M (2006) Key enzymes for flavour formation by lactic acid bacteria. Aust J Dairy Technol 61:88–96Google Scholar
  6. 6.
    Gómez de Cadiñanos LP, García-Cayuela T, Yvon M, Martínez-Cuesta MC, Peláez C, Requena T (2013) Inactivation of the panE gene in Lactococcus lactis enhances formation of cheese aroma compounds. Appl Environ Microbiol 79:3503–3506CrossRefGoogle Scholar
  7. 7.
    Yvon M, Rijnen L (2001) Cheese flavour formation by amino acid catabolism. Int Dairy J 11:185–201CrossRefGoogle Scholar
  8. 8.
    Yvon M, Thirouin S, Rijnen L, Fromentier D, Gripon JC (1997) An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavour compounds. Appl Environ Microbiol 63:414–419Google Scholar
  9. 9.
    Engels W, Alting AC, Arntz MMTG, Gruppen H, Voragen AGJ, Smit G, Visse S (2000) Partial purification and characterization of two aminotransferases from Lactococcus lactis subsp. cremoris B78 involved in the catabolism of methionine and branched chain amino acids. Int Dairy J 10:443–452CrossRefGoogle Scholar
  10. 10.
    Yvon M, Chambellon E, Bolotin A, Roudot Algaron F (2000) Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCD 763. Appl Environ Microbiol 66:571–577CrossRefGoogle Scholar
  11. 11.
    Yvon M, Berthelot S, Gripon JC (1998) Adding α-ketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. Int Dairy J 8:889–898CrossRefGoogle Scholar
  12. 12.
    Banks JM, Yvon M, Gripon JC, de la Fuente MA, Brechany EY, Williams AG, Muir DD (2001) Enhancement of amino acid catabolism in Cheddar cheese using α-ketoglutarate: amino acid degradation in relation to volatile compounds and aroma character. Int Dairy J 11:235–243CrossRefGoogle Scholar
  13. 13.
    Williams AG, Noble J, Banks JM (2004) The effect of α-ketoglutaric acid on amino acid utilization by nonstarter Lactobacillus spp. isolated from Cheddar cheese. Lett Appl Microbiol 38:289–295CrossRefGoogle Scholar
  14. 14.
    Van de Bunt B, Bron PA, Sijtsma L, De Vos WM, Hugenholtz J (2014) Use of non-growing Lactococcus lactis cell suspensions for production of volatile metabolites with direct relevance for flavour formation during dairy fermentations. Microb Cell Fact 13:176–185CrossRefGoogle Scholar
  15. 15.
    Smit G, van Hylckama JET, Engels WJM, Meijer L, Wouters JTM, Smit G (2005) Identification cloning and characterization of a Lactococcus lactis branched-chain α-keto acid decarboxylase involved in cheese flavour formation. Appl Environ Microbiol 71:303–311CrossRefGoogle Scholar
  16. 16.
    Smith EL, Austen BM, Bluementhal KM, Nyk JF (1975) Glutamate dehydrogenases. In: Boyer PD (ed) The enzymes. Academic Press, New York, pp 293–367Google Scholar
  17. 17.
    Tanous C, Kieronckyk A, Helinck S, Chambellon E, Yvon M (2002) Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains. Antonie Van Leeuwenhoek 82:271–278CrossRefGoogle Scholar
  18. 18.
    Morishita T, Yajima M (1995) Incomplete operation of biosynthetic and bioenergetic functions of the citric acid cycle in multiple auxotrophic lactobacilli. Biosci Biotechnol Biochem 59:251–255CrossRefGoogle Scholar
  19. 19.
    Peralta GH, Bergamini CV, Hynes ER (2016) Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci. Braz J Microbiol 477:41–748Google Scholar
  20. 20.
    Siragusa S, Fontana C, Cappa F, Caputo L, Cocconcelli PS, Gobetti M, De Angelis M (2011) Disruption of the gene encoding glutamate dehydrogenase affects growth amino acids catabolism and survival of Lactobacillus plantarum UC1001. Int Dairy J 21:59–68CrossRefGoogle Scholar
  21. 21.
    Liu M, Nauta A, Francke Ch, Siezen R (2008) Comparative genomics of enzymes in flavor-forming pathways from amino acids in lactic acid bacteria. Appl Environ Microbiol 74:4590–4600CrossRefGoogle Scholar
  22. 22.
    Kieronczyk A, Skeie S, Langsrud T, Yvon M (2003) Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids. Appl Environ Microbiol 69:734–739CrossRefGoogle Scholar
  23. 23.
    Helinck S, Le Bars D, Moreau D, Yvon M (2004) Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids. Appl Environ Microbiol 70:3855–3861CrossRefGoogle Scholar
  24. 24.
    Ayad EHE (2008) Biodiversity of lactococci in flavour formation for dairy products innovation. Alex J Food Sci Tech 3:31–43Google Scholar
  25. 25.
    Fernández de Palencia P, de la Plaza M, Amárita F, Requena T, Peláez C (2006) Diversity of amino acid converting enzymes in wild lactic acid bacteria. Enzyme Microb Technol 38:88–93CrossRefGoogle Scholar
  26. 26.
    Gutierrez-Méndez N, Valenzuela-Soto E, González-Códiva AF, Vallejo-Córdoba B (2008) α-keto glutarate biosynthesis in wild and industrial strains of Lactococcus lactis. Lett Appl Microbiol 47:202–207CrossRefGoogle Scholar
  27. 27.
    Tanous C, Chambellon E, Yvon M (2007) Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity. Microbiology 153:1664–1675CrossRefGoogle Scholar
  28. 28.
    Tanous C, Chambellon E, Le Bars D, Delespaul G, Yvon M (2006) Glutamate dehydrogenase activity can be transmitted naturally to Lactococcus lactis strains to stimulate amino acid conversion to aroma compounds. Appl Environ Microbiol 72:1402–1409CrossRefGoogle Scholar
  29. 29.
    Fontecha J, Pelaéz C, Júarez M, Requena T, Gómez C, Ramos M (1990) Biochemical and microbiological characteristics of artisanal hard goat’s cheese. J Dairy Sci 73:1150–1157CrossRefGoogle Scholar
  30. 30.
    Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A (2001) The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11:731–753CrossRefGoogle Scholar
  31. 31.
    Anderson DG, McKay LL (1983) Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46:549–552Google Scholar
  32. 32.
    Tanous C, Gori A, Rijnen L, Chambellon E, Yvon M (2005) Pathways for alpha-ketoglutarate formation by Lactococcus lactis and their role in amino acid catabolism. I Dairy J 15:759–770CrossRefGoogle Scholar
  33. 33.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  34. 34.
    De la Plaza M, Rodriguez A, Fernández de Palencia P, Martinez-Cuesta MC, Peláez C (2006) Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis isolates. Lett Appl Microbiol 43:637–644CrossRefGoogle Scholar
  35. 35.
    Tanous C, Chambellon E, Sepulchre A, Yvon M (2005) The gene encoding the glutamate dehydrogenase in Lactococcus lactis is part of a remnant Tn3 transposon carried by a large plasmid. J Bacteriol 187:5019–5022CrossRefGoogle Scholar
  36. 36.
    Requena T, McKay LL (1993) Plasmid profiles and relationship to lactose/utilization proteinase activity in a lactococcal strain isolated from semi hard natural cheese. Milchwissenschaft 48:264–268Google Scholar
  37. 37.
    Siezen RJ, Renckens B, van Swam I, Peters S, van Kranenburg R, Kleerebezem M, De Vos WM (2005) Complete sequences of four plasmids of Lactococcus lactis subsp. cremoris SK11 reveal extensive adaptation to the dairy environment. Appl Environ Microbiol 71:8371–8382CrossRefGoogle Scholar
  38. 38.
    Bachmann H, Starrenburg MJC, Dijkstra A, Molenaar D, Kleerebezem M, Rademaker JLW, van Hylckama Vlieg JET (2009) Regulatory phenotyping reveals important diversity within the species Lactococcus lactis. Appl Environ Microbiol 75:5687–5694CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Luz P. Gómez de Cadiñanos
    • 1
  • Carmen Peláez
    • 1
  • M. Carmen Martínez-Cuesta
    • 1
  • Tomás García-Cayuela
    • 1
  • Teresa Requena
    • 1
  1. 1.Departamento de Biotecnología y Microbiología de AlimentosInstituto de Investigación en Ciencias de la Alimentación CIAL (CSIC)MadridSpain

Personalised recommendations