Esimbekova E N, Torgashina I G, Kratasyuk V A
Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036, Russia.
Biochemistry (Mosc). 2009 Jun;74(6):695-700. doi: 10.1134/s0006297909060157.
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 K(m) (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.
对发光细菌的一种偶联酶系统（NAD(P)H：FMN氧化还原酶和荧光素酶）的性质进行了研究。这些酶及其底物被固定在不同类型的聚合物凝胶中：淀粉（多糖）和明胶（多肽）。固定在淀粉凝胶中的酶实现了最大活性产率（100%）。与可溶性偶联酶系统相比，在两种固定化系统中均观察到K(m)（表观）增加。固定在淀粉和明胶凝胶中增加了NAD(P)H：FMN氧化还原酶和荧光素酶偶联酶系统对外部物理和化学因素影响的抗性。最佳pH范围向酸性和碱性区域均有扩展。对浓盐溶液和高温的抗性也增加了。固定在淀粉凝胶中的偶联酶系统（活化能为30 kJ/mol）具有最佳的热稳定性。固定化偶联酶系统可用于生产用于生物发光分析的稳定且高活性的试剂。
