Bacterial bioluminescence with flavinmononucleotide activated by N-methylimidazole

Siberian Branch Krasnoyarsk, Russia, E-mail biotech ibp.ru 

Purification of luciferase. Luciferase was isolated from the recombinant strain of Echerichia coli SL60 with lux A and B genes of Photobacterium leiognathi from the collection of the Institute of Biophysics (RAS, SB) and purified by ion-exchange chromatography.5 The purity of the luciferase according to Laemmli electrophoresis was 90-95%. 

Measurement of luciferase activity. This was conducted by the following two methods 1. Luciferase activity was determined by a flavin injection assay in which 500 pL of photoreduced flavin mononucleotide were rapidly mixed with 500 pL of a solution containing luciferase, tetradecanal (4.7 mM) and 20 mM of phosphate buffer at pH 7.0 2. NADH or DTT was added to the cuvette, containing luciferase, tetradecanal and phosphate buffer and FMN. Under these conditions long-lived luminescence stimulated by multiple turnovers of enzyme was observed. 

Values of the rate constants of inhibition of the first order reaction were determined as a tangent of the angle of declination on the plot of log of intensity versus time. The rate constant of inhibition of the second order reaction calculated in this way, 

The injection of activated photoreduced FMN into the reaction mixture does not produce luminescence. At the same time luciferase does not lose its catalytic activity. The addition of native photoreduced FMN to the same mixture resulted in light response with appropriate kinetic parameters. 

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BACTERIAL BIOLUMINESCENCE WITH FLAVINMONONUCLEOTIDE ACTIVATED BY N-METHYLIMIDAZOLE... 

Bacterial Bioluminescence with Flavinmononucleotide Activated by N-Methylimidazole 37... 

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