Siberian Federal University

Issue

Journal of Siberian Federal University. Biology. 2018 11 (2)

Authors

Kirillova, Maria A.; Esimbekova, Elena N.; Ranjan, Rajeev; Torgashina, Irina G.; Kratasyuk, Valentina A.

Contact information

Kirillova, Maria A.: Siberian Federal University 79 Svobodny, Krasnoyarsk, 660041, Russia; Esimbekova, Elena N.: Siberian Federal University 79 Svobodny, Krasnoyarsk, 660041, Russia; Institute of Biophysics SB RAS FRC “Krasnoyarsk Science Center SB RAS” 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia; ; Ranjan, Rajeev: Siberian Federal University 79 Svobodny, Krasnoyarsk, 660041, Russia; Torgashina, Irina G.: Siberian Federal University 79 Svobodny, Krasnoyarsk, 660041, Russia; Kratasyuk, Valentina A.: Siberian Federal University 79 Svobodny, Krasnoyarsk, 660041, Russia; Institute of Biophysics SB RAS FRC “Krasnoyarsk Science Center SB RAS” 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia

Keywords

NADH:FMN-oxidoreductase; bacterial luciferase; bacterial contamination; Escherichia coli; Escherichia coli

Abstract

Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5’-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FMN

Pages

174-180

Paper at repository of SibFU

https://elib.sfu-kras.ru/handle/2311/71720

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).