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Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN–oxidoreductase–luciferase

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Abstract

We have studied the effects of a gel-like environment on the characteristics of enzyme preparations based on the coupled enzyme system of luminous bacteria, NADH:FMN–oxidoreductase–luciferase, to design a stable immobilizing reagent for bioluminescent analysis. Natural polymers, gelatin and starch, were used to create a viscous, structured microenvironment. The stability of the coupled enzyme system to such physical and chemical environmental factors as temperature, pH, and ionic strength in gelatin and starch-containing media was examined. It was shown that both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under specific conditions. In particular, the enzymes’ activity is increased twofold in the presence of 1 and 5 % of gelatin at 20 °C and 25 °C, respectively (temperatures lower than the gel point). Also, the acceptable pH range of the coupled enzyme system expands into the alkaline region and becomes 6.8–8.1. Stabilization at low ionic strength (0.01–0.06 mol L−1) is observed. At the same time, microenvironments based on either gelatin or starch do not change the enzymes’ thermal inactivation rate constants in the temperature range from 25 to 43 °C. Finally, gelatin and starch are suitable for development of a reagent for immobilization of enzymes which would be stable and resistant to physical and chemical environmental conditions.

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Acknowledgments

The authors are grateful to Anna Orlova for her help with the experimental work.

Declaration of interest

The work was supported by the Program of the Government of Russian Federation “Measures to attract leading scientists to Russian educational institutions” (grant no. 11. G34.31.0058), the Russian Academy of Sciences (program “Molecular and Cell Biology”, grant no. 6.8), and the state contract between the Ministry of Education and Science and Siberian Federal University, no. 1762.

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Authors and Affiliations

  1. Laboratory of Bioluminescent Biotechnologies, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, pr. Svobodnyi 79, Krasnoyarsk, 660041, Russia

    Anna Bezrukikh, Elena Esimbekova, Elena Nemtseva, Valentina Kratasyuk & Osamu Shimomura

  2. Institute of Biophysics SB RAS, Akademgorodok 50/50, Krasnoyarsk, 660036, Russia

    Elena Esimbekova, Elena Nemtseva & Valentina Kratasyuk

Authors
  1. Anna Bezrukikh
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  2. Elena Esimbekova
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  3. Elena Nemtseva
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  4. Valentina Kratasyuk
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  5. Osamu Shimomura
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Corresponding author

Correspondence to Elena Esimbekova.

Additional information

Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli.

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Bezrukikh, A., Esimbekova, E., Nemtseva, E. et al. Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN–oxidoreductase–luciferase. Anal Bioanal Chem 406, 5743–5747 (2014). https://doi.org/10.1007/s00216-014-7987-1

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  • DOI: https://doi.org/10.1007/s00216-014-7987-1

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