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

Anal Bioanal Chem. 2014 Sep;406(23):5743-7. doi: 10.1007/s00216-014-7987-1. Epub 2014 Jul 8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aliivibrio fischeri / enzymology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • FMN Reductase / chemistry*
  • FMN Reductase / metabolism
  • Gelatin / chemistry
  • Kinetics
  • Luciferases / chemistry*
  • Luciferases / metabolism
  • Luminescent Measurements
  • Starch / chemistry

Substances

  • Bacterial Proteins
  • Gelatin
  • Starch
  • Luciferases
  • FMN Reductase