摘要
基于乙醇(EtOH)/乙醇脱氢酶(ADH)/氧化型辅酶I(NAD+)催化反应体系,建立了一种用于ADH活性测定的循环催化流动分析法(RCFA),并优选该测定体系的实验条件,得到如下参数:Tris-HCl缓冲液浓度(pH 8.9)为0.1 mol/L,EtOH的浓度为15.6 mol/L,NAD+浓度为9.0 mmol/L,ADH用量20μL/次,反应液流速为0.98 mL/min,反应液体积为2 mL;ADH的测定范围为0.6～50 U/L,检出限为0.14 U/L,相对标准偏差≤1.3%(n=11)。RCFA法具有测定过程简便、快速、自动化特点,能对ADH催化反应过程进行连续循环检测,可对ADH活性变化实现动态研究。RCFA法使反应液循环不但节省了大量的试剂和酶量、降低了测定成本,而且循环测定时不存在人为误差,提高了测定结果的准确度。
Based on the ethanol（EtOH） / alcohol dehydrogenase（ADH）/ oxidized form of nicotinamide-adenine dinucleotide（NAD＋） system,a recirculating-catalysis flow analytical method（RCFA） for the determination of ADH activity had been developed.The experimental conditions were optimized as follows： the concentration of Tris-HCl buffer（pH8.9） and EtOH were 0.1mol/L and 15.6 mol/L respectively,and the concentration of NAD＋ was 9.0 mmol/L,the use level of ADH for each determination was only 20 μL,the flow rate of the reaction mixture solution was 0.98 mL/min,and the reaction solution volume was 2 mL.The determination range of ADH was 0.6～50U/L,the detection limit was 0.14U/L,and the relative standard divation（RSD） was 2.0 %（n=11）.RCFA had the advantages including simple operation and rapid determination and automated analysis and it could continuously monitor the enzymatic reaction process and the dynamic performance of ADH.In addition,the recircu-lating of the reaction mixture solution could not only save large amount of reagents and enzyme（reducing determination costs） but also improve determination accuracy because there was no personal error in the determination process.
出处
《酿酒科技》
2010年第10期100-102,114,共4页
Liquor-Making Science & Technology
关键词
循环催化流动分析
乙醇脱氢酶
乙醇
氧化型辅酶I
酿酒酵母菌
recirculating-catalysis flow analysis
alcohol dehydrogenase
ethanol
oxidized form of nicotinamide-adenine dinucleotide
Saccharomyces cerevisiae
