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Comparative Study of Immobilized and Soluble NADH:FMN-Oxidoreductase–Luciferase Coupled Enzyme System

E. N. Esimbekova1,2*, I. G. Torgashina2, and V. A. Kratasyuk1,2

1Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Akademgorodok, 660036 Krasnoyarsk, Russia; fax: (3912) 433-400; E-mail: Esimbekova@yandex.ru

2Siberian Federal University, pr. Svobodnyi 79, 660041 Krasnoyarsk, Russia; fax: (3912) 448-781; E-mail: VKratasyuk@yandex.ru

* To whom correspondence should be addressed.

Received March 23, 2008; Revision received July 7, 2008
The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in Km app was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.
KEY WORDS: bioluminescence, immobilization, thermostability, luciferase, starch, gelatin

DOI: 10.1134/S0006297909060157