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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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.
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
- Bacterial luciferase
- Stabilization of enzymes