Biochemistry (Moscow)

, Volume 74, Issue 6, pp 695–700 | Cite as

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system

  • E. N. EsimbekovaEmail author
  • I. G. Torgashina
  • V. A. Kratasyuk


The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K m app was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.

Key words

bioluminescence immobilization thermostability luciferase starch gelatin 



myristic aldehyde


NADH:FMN-oxidoreductase-luciferase coupled enzyme system


multicomponent immobilized reagent including the NADH:FMN-oxidoreductase-luciferase coupled enzyme system, myristic aldehyde, and NADH


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kratasyuk, V. A., and Gitelson, I. I. (1987) Usp. Mikrobiol., 21, 3–30.Google Scholar
  2. 2.
    Roda, A., Pasini, P., Mirasoli, M., Michelini, E., and Guardigli, M. (2004) Trends Biotechnol., 22, 295–303.PubMedCrossRefGoogle Scholar
  3. 3.
    Rozhko, T. V., Kudryasheva, N. S., Aleksandrova, M. A., Bondareva L. G., Bolsunovsky, A. Ya., and Vydryakova, G. V. (2008) J. Siber. Fed. Univ. Biol., 1, 60–65.Google Scholar
  4. 4.
    Kudryasheva, N. S. (2006) J. Photochem. Photobiol. B, 83, 77–86.PubMedCrossRefGoogle Scholar
  5. 5.
    Hastings, J. W., and Johnson, C. H. (2003) Meth. Enzymol., 360, 75–105.PubMedCrossRefGoogle Scholar
  6. 6.
    Petushkov, V. N., Kratasyuk, G. A., Kratasyuk, V. A., and Belobrov, P. I. (1982) Biokhimiya, 47, 1773–1777.Google Scholar
  7. 7.
    Kuznetsov, A. M., Tyul’kova, N. A., Kratasyuk, V. A., Abakumova, V. V., and Rodicheva, E. K. (1997) Sibir. Ekol. Zh., 5, 459–465.Google Scholar
  8. 8.
    Kricka, L., Wienhausen, G., Hinkley, J., and DeLuca, M. (1983) Anal. Biochem., 129, 392–397.PubMedCrossRefGoogle Scholar
  9. 9.
    Oda, K., Yoshida, S., Hirose, S., and Takeda, T. (1989) Clin. Chem. Acta, 185, 17–24.CrossRefGoogle Scholar
  10. 10.
    Ugarova, N., and Lebedeva, O. (1987) Appl. Biochem. Biotechnol., 15, 35–51.PubMedCrossRefGoogle Scholar
  11. 11.
    Kratasyuk, V., and Esimbekova, E. (2003) in Polymeric Biomaterials. The PBM Series, Vol. 1 (Arshady, R., ed.) Citus Books, London, pp. 301–343.Google Scholar
  12. 12.
    Esimbekova, E., and Kratasyuk, V. (2005) in Bioluminescence & Chemiluminescence: Progress & Perspectives (Tsuji, A., Matsumoto, M., Maeda, M., Kricka, L., and Stanley, P., eds.) World Scientific Publishing Co., Singapore, pp. 237–240.CrossRefGoogle Scholar
  13. 13.
    RF Patent (2005) No. 2252963.Google Scholar
  14. 14.
    Esimbekova, E. N., Kratasyuk, V. A., and Torgashina, I. G. (2007) Enzyme Microb. Technol., 40, 343–346.CrossRefGoogle Scholar
  15. 15.
    Kratasyuk, V. A., Abakumova, V. V., and Kim, N. B. (1994) Biochemistry (Moscow), 59, 761–766.Google Scholar
  16. 16.
    Berezin, I. V. (ed.) (1976) Immobilized Enzymes [in Russian], Vol. 2, Moscow State University Publishers, Moscow.Google Scholar
  17. 17.
    James, T. H. (1980) in The Theory of Photographic Process [Russian translation], Khimiya, Leningrad, pp. 55–70.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • E. N. Esimbekova
    • 1
    • 2
    Email author
  • I. G. Torgashina
    • 2
  • V. A. Kratasyuk
    • 1
    • 2
  1. 1.Institute of BiophysicsSiberian Branch of the Russian Academy of SciencesKrasnoyarskRussia
  2. 2.Siberian Federal UniversityKrasnoyarskRussia

Personalised recommendations