NADH依赖型酶活性检测方法的建立

An Efficient and Economic Method for Detecting the Activity of NADH Dependent Enzyme

目的:建立一种NADH依赖型酶活性检测的方法。方法:将FDH、LeuDH串联克隆到表达载体pET-22b(+)中,转化至E.coli,并向培养液中分别添加去离子水、甲酸胺、三甲基丙酮酸,反应一段时间后,检测NADH的吸光度。同时通过测定氨气的产生判断FDH活性;通过薄层层析检测判断LeuDH活性;比较NADH吸光度测定结果与常规方法结果是否一致。结果:通过测定氨气的产生,证明FDH具有活性;此时NADH吸光度上升亦说明FDH具有活性;两种方法结论一致。薄层层析检测,生成叔亮氨酸,证明LeuDH具有活性;此时NADH吸光度下降亦说明LeuDH具有活性;两种方法结论一致。结论:通过检测菌体内部NADH吸光度的变化检测NADH依赖型酶活性的方法可行。

Objective： To establish a NADH depending enzyme activity detection method.Methods： FDH and LeuDH were co-expressed in E.coli and appropriate substrate was added;the FDH activity was measured by NH3 production and the LeuDH activity measured by TLC analysis,which were also detected by measuring the NADH absorbance.The results from both methods were compared.Results： Both NH3 production and the NADH absorbance detection proved FDH activity;at the same time,tertiary leucine production and the NADH absorbance detection proved LeuDH activity.Conclusion： The detection of NADH absorbance changes could be employed in measuring the absorbance of NADH.