Bezrukikh Anna, Esimbekova Elena, Nemtseva Elena, Kratasyuk Valentina, Shimomura Osamu
Laboratory of Bioluminescent Biotechnologies, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, pr. Svobodnyi 79, Krasnoyarsk, 660041, Russia.
Anal Bioanal Chem. 2014 Sep;406(23):5743-7. doi: 10.1007/s00216-014-7987-1. Epub 2014 Jul 8.
We have studied the effects of a gel-like environment on the characteristics of enzyme preparations based on the coupled enzyme system of luminous bacteria, NADH:FMN-oxidoreductase-luciferase, to design a stable immobilizing reagent for bioluminescent analysis. Natural polymers, gelatin and starch, were used to create a viscous, structured microenvironment. The stability of the coupled enzyme system to such physical and chemical environmental factors as temperature, pH, and ionic strength in gelatin and starch-containing media was examined. It was shown that both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under specific conditions. In particular, the enzymes' activity is increased twofold in the presence of 1 and 5% of gelatin at 20 °C and 25 °C, respectively (temperatures lower than the gel point). Also, the acceptable pH range of the coupled enzyme system expands into the alkaline region and becomes 6.8-8.1. Stabilization at low ionic strength (0.01-0.06 mol L(-1)) is observed. At the same time, microenvironments based on either gelatin or starch do not change the enzymes' thermal inactivation rate constants in the temperature range from 25 to 43 °C. Finally, gelatin and starch are suitable for development of a reagent for immobilization of enzymes which would be stable and resistant to physical and chemical environmental conditions.
我们研究了凝胶状环境对基于发光细菌、NADH：FMN氧化还原酶-荧光素酶的偶联酶系统的酶制剂特性的影响，以设计一种用于生物发光分析的稳定固定试剂。天然聚合物明胶和淀粉被用于创建一个粘性的、结构化的微环境。研究了偶联酶系统在含明胶和淀粉的介质中对温度、pH值和离子强度等物理和化学环境因素的稳定性。结果表明，在特定条件下，明胶和淀粉对发光细菌的酶都有稳定作用。特别是，在20℃和25℃时，分别在1%和5%的明胶存在下（温度低于凝胶点），酶的活性增加了两倍。此外，偶联酶系统可接受的pH范围扩展到碱性区域，变为6.8-8.1。在低离子强度（0.01-0.06 mol L(-1)）下观察到稳定作用。同时，基于明胶或淀粉的微环境在25至43℃的温度范围内不会改变酶的热失活速率常数。最后，明胶和淀粉适用于开发一种稳定且能抵抗物理和化学环境条件的酶固定试剂。
