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This review describes the principle and applications of bioluminescent enzymatic toxicity bioassays. This type of assays uses bacterial coupled enzyme systems: NADH:FMNoxidoreductase and luciferase to replace living organisms in developing cost-competitive biosensors for environmental, medical and industrial applications. These biosensors instantly signal chemical and biological hazards and allow for detecting a great amount of toxic compounds with advantages associated with fast results, high sensitivity, simplicity, low cost and safety of the procedure.

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.

: This review examines the general principles of bioluminescent enzymatic toxicity bioassays and describes the applications of these methods and the implementation in commercial biosensors. Bioluminescent enzyme system technology (BEST) has been proposed in the bacterial coupled enzyme system, wherein NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. For widespread use of BEST, the multicomponent reagent "Enzymolum" has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase, and their substrates, co-immobilized in starch or gelatin gel. Enzymolum is the central part of Portable Laboratory for Toxicity Detection (PLTD), which consists of a biodetector module, a sampling module, a sample preparation module, and a reagent module. PLTD instantly signals chemical-biological hazards and allows us to detect a wide range of toxic substances. Enzymolum can be integrated as a biological module into the portable biodetector-biosensor originally constructed for personal use. Based on the example of Enzymolum and the algorithm for creating new enzyme biotests with tailored characteristics, a new approach was demonstrated in biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. Possible applications of enzyme bioassays are seen in the examples for medical diagnostics, assessment of the effect of physical load on sportsmen, analysis of food additives, and in practical courses for higher educational institutions and schools. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 min), high sensitivity, simplicity and safety of procedure, and possibility of automation of ecological monitoring; the required luminometer is easily available.

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