细菌荧光素酶体外发光体系的建立及应用于NADH定量检测

Quantitative detection of NADH by in vitro bacterial luciferase bioluminescent

【目的】本研究旨在建立鳆发光杆菌(Photobacterium leiognathi)YL荧光素酶:FMN-NADH氧化还原酶体外发光双酶体系,并对荧光素酶:FMN-NADH氧化还原酶体外发光双酶体系应用于NADH的定量检测进行初步探索。【方法】利用从鳆发光杆菌提取并经部分纯化的荧光素酶和FMN-NADH氧化还原酶,通过优化体系中各底物的添加量,实现荧光素酶的体外发光。【结果】荧光素酶:FMN-NADH氧化还原酶体外发光双酶体系为:1mL酶液中添加100μL十二烷醛(27mmol/L)、0.5μLFMN-Na(10mmol/L)、300μLNADH(0.14mmol/L)。NADH与荧光素酶:FMN-NADH氧化还原酶体系的发光强度呈良好的线性关系,其线性范围为1.0×10-10～1.0×10-8mol/L。【结论】荧光素酶:FMN-NADH氧化还原酶体外发光双酶体系可以简便、灵敏、快速的定量检测NADH,为其进一步应用于环境检测、食品卫生与安全等领域活细菌数量的检测奠定了基础。

[ Objective] The study aimed at establishing the bacterial luciferase： FMN-NADH oxidoreductase bioluminescent system in vitro and evaluating its potential for quantitative detection of NADH. [ Methods ] By optimizing the amount of substrates, we set up the coupled luciferase： FMN-NADH oxidoreductase bioluminescent system in vitro, based on the crude extract from Photobacteriurn leiognathi YL. [Results] The in vitro coupled bacterial luciferase： FMN-NADH oxidoreductase bioluminescent system was： 1 mL crude extract, 27 mmol/L Dodecane 100 μL, 10 mmol/L FMN-Na 0.5 μL and 0.14 mmol/L NADH 300 μL. Furthermore, we developed a method for quantitative detection of NADH according to the bioluminescence of NADH catalyzed of bacterial luciferase： FMN-NADH oxidoreductase system in vitro. A good linear relationship of NADH concentration was in a range of 1.0 × 10^-10 to 1.0 × 10^-8 mol/L. [ Conclusion] The bacterial luciferase： FMN-NADH oxidoreductase system used to measure NADH concentration was a good attempt to detect living bacterial cells in the fields of environment, food sanitation and other related.