厌氧发酵有机酸体系中NAD+和NADH测定方法的建立

Establishment of the determination of NAD+ and NADH in the anaerobic fermentation of organic acids

建立一种酶法高效测定厌氧发酵有机酸体系中NAD+和NADH的方法。利用酶循环机理,以1.0 mol/L Bicine buffer(pH8.0)、纯乙醇、40 mmol/L EDTA(pH8.0)、4.2 mmol/L MTT、16.6mmol/L PES和50μL的乙醇脱氢酶作为混合反应体系,采用紫外可见分光光度计在570 nm处测定吸光值变化情况,能将发酵体系中的NAD+和NADH准确定量,检出限分别为1.3×10-6 mol/L和5.9×10-6 mol/L,回收率分别在94%～98%和96%～101%范围内。能够快速、精确测定厌氧发酵体系中的NAD+和NADH含量,对于指导厌氧发酵体系辅酶代谢调控分析具有重要意义。

An enzyme method for determining NAD^＋ and NADH highly efficient in the anaerobic fermentation broth was developed. Using the recycling mechanism, 1.0 mol/L Bicine buffer（pH8.0）, pure ethanol, 40 mmol/L EDTA（pH8.0）, 4.2 mmol/L MTT, 16.6 mM PES and 50 μL alcohol dehydrogenase as mixed reaction system, and determining the change of absorbance at the wavelength of 570 nm in UV-Vis spectrophotometer could quantitate NAD^＋ and NADH of fermentation system exactly. The detection limit was 5.9×10^-7 mol/L and 1.3×10^-7 mol/L, and the recovery were 94%-98% and 96%-101% respectively. The contents of NAD^＋ and NADH in the anaerobic fermentation broth could be rapidly and exactly determined by this method. It is very useful for the anaerobic metabolic regulation analysis.