Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus

  • 307 Accesses

  • 36 Citations

Abstract

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to α-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70°C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25°C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45°C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Alen N, Steen IH, Birkeland N-K, Lien T (1997) Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus. Arch Microbiol 168:536–539

  2. Basu AK, Chattopadhyay P, Roychudhuri U, Chakraborty R (2006) A biosensor based on co-immobilized L-glutamate oxidase and L-glutamate dehydrogenase for analysis of monosodium glutamate in food. Biosens Bioelectron 21:1968–1972

  3. Bertocchi P, Compagnone D, Palleschi G (1996) Amperometric ammonium ion and urea determination with enzyme-based probes. Biosens Bioelectron 11:1–10

  4. Betancor L, López-Gallego F, Hidalgo A, Fuentes M, Podrasky O, Kuncova G, Guisán JM, Fernández-Lafuente R (2005) Advantages of the pre-immobilization of enzymes on porous supports for their entrapment in sol–gels. Biomacromolecules 6:1027–1030

  5. Blanco RM, Guisán JM (1989) Stabilization of enzymes by multipoint covalent attachment to agarose–aldehyde gels. Borohydride reduction of trypsin–agarose derivatives. Enzyme Microb Technol 11:360–366

  6. Bolivar JM, Wilson L, Ferrarotti SA, Fernandez-Lafuente R, Guisan JM, Mateo C (2006a) Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports. Biomacromolecules 7:669–673

  7. Bolivar JM, Wilson L, Ferrarotti SA, Guisan JM, Fernandez-Lafuente R, Mateo C (2006b) Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose. J Biotechnol 125:85–94

  8. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

  9. Britton KL, Baker PJ, Borgers KMM, Engel PC, Pasquo A, Rice DW, Robb FT, Scandurra R, Stillman TJ, Yip KSP (1995) Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litorales. Eur J Biochem 229:688–695

  10. Chenault HK, Simon ES, Whitesides GM (1988) Cofactor regeneration for enzyme-catalysed synthesis. Biotechnol Genet Eng Res 6:221–245

  11. Cleland WW (1967) The statistical analysis of enzyme kinetic data. Adv Enzymol Relat Areas Mol Biol 29:1–32

  12. Cleland WW (1977) Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv Enzymol Relat Areas Mol Biol 45:273–387

  13. Cowan DA (1997) Thermophilic proteins: stability and function in aqueous and organic solvents. Comp Biochem Phys A 118:429–438

  14. Daniel RM, Cowan DA (2000) Biomolecular stability and life at high temperatures. Cell Mol Life Sci 57:250–264

  15. Díaz S, Pérez-Pomares F, Pire C, Ferrer J, Bonete MJ (2006) Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei. Extremophiles 10:105–115

  16. Doong RA, Shih HM (2006) Glutamate optical biosensor based on the immobilization of glutamate dehydrogenase in titanium dioxide sol-gel matrix. Biosens Bioelectron 22:185–191

  17. Eckstein M, Daubmann T, Kragl U (2004) Recent developments in NAD(P)H regeneration for enzymatic reductions in one and two phase systems. Biocatal Biotransform 22:89–96

  18. Fernández-Lafuente R, Hernández-Jústiz O, Mateo C, Fernández-Lorente G, Terreni M, Alonso J, Garcia-López JL, Moreno MA, Guisán JM (2001) Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions. Biomacromolecules 2:95–104

  19. Guisán JM (1988) Aldehyde–agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb Technol 10:375–382

  20. Gupta MN (1991) Thermostabilization of proteins. Biotechnol Appl Biochem 14:1–11

  21. Ha JS, Kim K, Song JJ, Bae JW, Lee SG, Lee SC, Poo H, Shin CS, Sung MH (2003) Thermostable glutamate dehydrogenase from a commensal thermophile, Symbiobacterium toebii overproduction, characterization, and application. J Mol Catal B: Enzym 26:231–240

  22. Higuchi S, Kobayashi T, Kimura K, Horikoshi K, Kudo T (1997) Molecular cloning, nucleotide sequence and expression in Escherichia coli of hyperthermophilic glutamate dehydrogenase gene from Thermococcus profundus. J Fermen Bioeng 83:405–411

  23. Hudson RC, Ruttersmith LD, Daniel RM (1993) Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1. Biochim Biophys Acta 1202:244–250

  24. Hummel W (1997) New alcohol dehydrogenases for the synthesis of chiral compounds. Adv Biochem Eng Biotechonol 58:145–184

  25. Hummel W (1999) Large-scale applications of NAD(P)-dependent oxidorreductases: recent developments. Trends Biotechnol 17:487–492

  26. Jaenicke R, Schurig H, Beaucamp N, Ostendorp R (1996) Structure and stability of hyperstable proteins: glycolytic enzymes from hyperthermophilic bacterium Thermotoga maritime. Adv Prot Chem 48:181–269

  27. Joe A, Murray CS, McBride BC (1994) Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant. Infect Immun 62:1358–1368

  28. Klibanov AM (1979) Enzyme stabilization by immobilization. Anal Biochem 93:1–25

  29. Klibanov AM (1983) Approaches to enzyme stabilization. Biochem Soc Trans 11:19–20

  30. Knapp S, de Vos WM, Rice D, Ladenstein R (1997) Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima at 3.0 Å resolution. J Mol Biol 267:916–932

  31. Kobayashi T, Higuchi S, Kimura K, Kudo T, Horikoshi K (1995) Properties of glutamate dehydrogenase and its involvement in alanine production in a hyperthermophilic archaeon, Thermococcus profundus. J Biochem 118:587–592

  32. Kort R, Liebl W, Labedan B, Forterre P, Eggen RIL, de Vos WM (1997) Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications. Extremophiles 1:52–60

  33. Kujo C, Ohshima T (1998) Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents. Appl Environ Microbiol 64:2152–2157

  34. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685

  35. Lasa I, de Grado M, de Pedro MA, Berenguer J (1992) Development of Thermus–Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus. J Bacteriol 174:6424–6431

  36. Lebbink JHG, Knapp S, van der Oost J, Rice D, Ladenstein R, de Vos WM (1998) Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region. J Mol Biol 280:287–296

  37. Lebbink JHG, Kengen SWM, Oost JVD, de Vos WM (1999) Glutamate dehydrogenase from hyperthermophilic Bacteria and Archaea: determinants of thermostability and catalysis at extremely high temperatures. J Mol Catal B Enzym 7:133–145

  38. Leonida MD (2001) Redox enzymes used in chiral syntheses coupled to coenzyme regeneration. Curr Med Chem 8:345–369

  39. Liu W, Wang P (2007) Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol Adv 25:369–384

  40. Maras B, Consalvi V, Chiaraluce R, Politi L, de Rosa M, Scandurra R (1992) The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-e-methyllysine related to thermostability? Eur J Biochem 203:81–87

  41. Maras B, Valiante S, Chiaraluce R, Consalvi V, Politi L, de Rosa M, Bossa F, Scandurra R, Barra D (1994) The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium. J Prot Chem 13:253–259

  42. Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, Palomo JM, Grazú V, Pessela BCC, Giacomini C, Irazoqui G, Villarino A, Ovsejevi A, Batista-Viera F, Fernandez-Lafuente R, Guisán JM (2005) Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb Technol 37:456–462

  43. Mateo C, Palomo JM, Fuentes M, Betancor L, Grazu V, Lopez-Gallego F, Pessela BCC, Hidalgo A, Fernandez-Lorente G, Fernandez-Lafuente R, Guisan JM (2006) Glyoxyl-agarose: a fully inert hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb Technol 39:274–280

  44. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40:1451–1463

  45. Molitor M, Dahl C, Molitor I, Schafer U, Speich N, Huber R, Deutzmann R, Truper HG (1998) A dissimilatory sirohaem-sulfite-reductase-type protein from the hyperthermophilic archaeon Pyrobaculum islandicum. Microbiology 144:529–541

  46. Nakkharat P, Haltrich D (2006) Lactose hydrolysis and formation of galactooligosaccharides by a novel immobilized b-galactosidase from the thermophilic fungus Talaromyces thermophilus. Appl Biochem Biotechnol 129:215–225

  47. Nawani N, Singh R, Kaur J (2006) Immobilization and stability studies of a lipase from thermophilic Bacillus sp: the effect of process parameters on immobilization of enzyme. Electron J Biotechnol 9:559–565

  48. Owusu RK, Cowan DA (1989) Correlation between microbial protein thermostability and resistance to denaturation in aqueous: organic solvent two-phase systems. Enzyme Microb Technol 11:568–574

  49. Pedroche J, Yust MM, Mateo C, Fernández-Lafuente R, Girón-Calle J, Alaiz M, Vioque J, Guisán JM, Millán F (2007) Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: correlation between enzyme–support linkages and thermal stability. Enzyme Microb Technol 40:1161–1167

  50. Pessela BCC, Mateo C, Carrascosa AV, Vian A, García JL, Rivas G, Alfonso C, Guisan JM, Fernández-Lafuente R (2003) One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-his-tagged b-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate–Epoxy sepabeads. Biomacromolecules 4:107–113

  51. Rahman RNZA, Fujiwara S, Takagi M, Imanaka T (1998) Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDH. Mol Gen Genet 257:338–247

  52. Reitzer LJ, Magasanik B (1987) Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, l-alanine, and d-alanine. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 302–320

  53. Robb FT, Maeder DL, DiRuggiero J, Borges KM, Tolliday N (2001) Glutamate dehydrogenases from hyperthermophiles. Methods Enzymol 331:26–41

  54. Rodriguez BB, Bolbot JA, Tothill IE (2004a) Urease–glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples. Anal Bioanal Chem 380:284–292

  55. Rodriguez BB, Bolbot JA, Tothill IE (2004b) Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples. Biosens Bioelectron 19:1157–1167

  56. Ruiz JL, Ferrer J, Camacho M, Bonete M (1998) NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties. FEMS Microbiol Lett 159:15–20

  57. Ruiz JL, Ferrer J, Pire C, Llorca FI, Bonete MJ (2003) Denaturation studies by fluorescence and quenching of thermophilic protein NAD+–glutamate dehydrogenase from Thermus thermophilus HB8. J Prot Chem 22:295–301

  58. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, New York

  59. Smith EL, Austen BM, Blumenthal KM, Nyc JF (1975) Glutamate dehydrogenases. In: Boyer PD (ed) The enzymes. Academic, New York, pp 293–367

  60. Tardioli PW, Zanin GM, de Moraes FF (2006) Characterization of Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on glyoxyl-agarose. Enzyme Microb Technol 39:270–1278

  61. Van der Donk W, Zhao H (2003) Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol 14:421–426

  62. Wandrey C (2004) Biochemical reactions engineering for redox reactions. Chem Rev 4:254–265

  63. Yip KSP, Britton KL, Stillman TJ, Lebbink JHG, de Vos WM, Robb FT, Rice DW (1998) Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family. Eur J Biochem 255:336–346

  64. Zhao H, Van der Donk W (2003) Regeneration of cofactors for use in biocatalysis. Curr Opin Biotechnol 14:583–589

Download references

Acknowledgments

This work has been supported by grants of code S0505/PPQ/0344 of the Comunidad Autónoma de Madrid (CAM) to J. Berenguer and J.M. Guisán and grants BIO2007-60245 (J. Berenguer) and CTQ2005-02420/PPQ (R. Fernández-Lafuente) from the Ministry of Education and Science (MEC). A Ramón y Cajal Contract, MEC (César Mateo). An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged. J.M. Bolívar and J.Rocha-Martín are the holders of a Ph.D. fellowship from CAM, and F. Cava holds a contract from the MEC. We thank A. Berenguer for his help during the writing of this paper.

Author information

Correspondence to Jose M. Guisán.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bolivar, J.M., Cava, F., Mateo, C. et al. Immobilization–stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus . Appl Microbiol Biotechnol 80, 49 (2008). https://doi.org/10.1007/s00253-008-1521-3

Download citation

Keywords

  • Redox enzymes
  • Thermophilic enzymes hyperstabilization
  • Multipoint immobilization
  • Multimeric enzyme stabilization
  • Cofactor regeneration
  • Glutamate biosensor