Ecology & Safety ISSN 1314-7234, Volume 12, 2018 Journal of International Scientific Publications www.scientific-publications.net Page 135 APPLICATION OF BIOLUMINESCENT ENZYMATIC TESTS IN ECOTOXICOLOGY Elena N. Esimbekova 1,2 *, Valeriya P. Kalyabina 2,1 , Valentina A. Kratasyuk 2,1 1 Institute of Biophysics SB RAS, Federal Research Center ‘Krasnoyarsk Science Center SB RAS’, Akademgorodok 50/50, Krasnoyarsk 660036, Russia 2 Siberian Federal University, pr. Svobodnii 79, Krasnoyarsk 660041, Russia Abstract This paper 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 wide- spread use of BEST, the multi-component reagent ‘Enzymolum’ has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel. ‘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 of 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. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 minutes), high sensitivity, simplicity and safety of procedure, possibility of automation of ecological monitoring; required luminometer is easily available. Keywords: bioluminescence, luciferase, enzymatic assay, immobilization, integral water toxicity, ecological monitoring 1. INTRODUCTION Historically, the application of bacterial luminescence in toxicology began with the usage of luminous bacteria for ecological monitoring and they are still widely used (Girotti et al., 2008; Roda et al., 2009; Fernández-Piñas et al., 2014; Xu et al., 2014). These methods made it possible to determine environmental pollution by comparing the light emission intensity of luminous bacteria in control with samples. As opposed to other test objects such as paramecia, algae, crustaceans, and so on, the bioluminescent assay is faster (typically < 30 min). However, as with other living organisms, living luminous bacteria is petulant. The failure to maintain the stable state of bacterial culture during measurements and storage results in low accuracy of measurement, a clear disadvantage of this method caused by the ‘‘petulance’’. The bacteria react to the appearance of toxic substances either by decreasing or by increasing the luminous intensity, often leading to ambiguous interpretation of results. Because of these shortcomings the assay based on luminous bacteria didn’t show very good results in ecological laboratories. To overcome those difficulties it was suggested to use enzymes of luminous bacteria NAD(P)H:FMN-oxidoreductase and luciferase in soluble and immobilized forms (Kratasyuk, 1990; Esimbekova et al., 2013). Since 1990, bioluminescent enzymatic toxicity assay has been developed (Kratasyuk, 1990), and is nowadays actively used in ecology, medicine, agriculture, and other areas (Esimbekova et al., 2014; Kratasyuk and Esimbekova, 2015).