巨大芽孢杆菌产亚硝酸还原酶活性测定条件优化

Optimization of determination conditions of nitrite reductase activity produced by Bacillus megaterium

为探明巨大芽胞杆菌产亚硝酸还原酶(nitrite reductase,NiR)的催化特性,更好发挥其催化性能,该文从缓冲液种类、电子供体种类及浓度、NaCl浓度、加酶量以及反应温度和反应时间等几个方面研究了巨大芽胞杆菌产亚硝酸盐还原酶活性测定的适宜条件。研究结果表明,该菌产NiR酶活测定体系为:在250μL反应体系中加入Na2HPO(40.1mol/L)-柠檬酸(0.05mol/L)缓冲液(pH6.0)125μL;1mol/L NaCl 15μL;0.05mol/L甲基紫精(MV)7.5μL、0.05mol/L硫代硫酸钠20μL和0.15mol/L连二亚硫酸钠20μL作为电子供体,加酶量50μL;优化后的反应条件为反应温度35℃,反应时间1.5min。

In order to investigate and utilize the catalytic characteristics of nitrite reductase （NiR） produced by Bacillus megatenum, the determination conditions of NiR activity were optimized by buffers, electron donors and their concentrations, NaC1 concentration, enzyme dosage, reaction temperature and time. The determination system（250μL）of NiR activity consisted of 0.1 mol/L Na2HPO4-0.05mol/L citric acid buffer（pH6.0）125μL, 1 mol/L NaCl 15μL, 3 electron donors including 0.05mol/L methyl viologen 7.5μL, 0.05mol/L sodium thiosulfate 201μL and 0.15mol/L sodium dithionite 20μL, and enzyme 50μL. The reaction was performed at 35℃ for 1.5min.