title: Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN-oxidoreductase-luciferase.
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.
authors.affiliation: Laboratory of Bioluminescent Biotechnologies, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, pr. Svobodnyi 79, Krasnoyarsk, 660041, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Bezrukikh
authors.forename: Elena
authors.initials: E
authors.lastname: Esimbekova
authors.forename: Elena
authors.initials: E
authors.lastname: Nemtseva
authors.forename: Valentina
authors.initials: V
authors.lastname: Kratasyuk
authors.forename: Osamu
authors.initials: O
authors.lastname: Shimomura
mesh_terms: D048908:Aliivibrio fischeri / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D038181:FMN Reductase / Q000737:chemistry* / Q000378:metabolism; D005780:Gelatin / Q000737:chemistry; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements; D013213:Starch / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005780:Gelatin; D013213:Starch; D008156:Luciferases; D038181:FMN Reductase