工程菌产酶制备GABA测定条件的优化  

Optimization the determination conditions of γ-aminobutyric acid producting by recombinant E.coli glutamate decarboxylase

作　　者：张珂[1] 薛正莲[1] 

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000

出　　处：《安徽工程大学学报》2012年第1期29-31,35,共4页Journal of Anhui Polytechnic University

摘　　要：基于Berthelot显色反应,采用正交设计法优化了测定大肠杆菌工程菌谷氨酸脱羧酶转化L-谷氨酸生成γ-氨基丁酸含量的条件.实验确定的最佳测定条件为:2.0mL 0.2mol/L pH6.0的醋酸缓冲液(内含0.1mmol/L的PLP,0.2mol/L的L-谷氨酸,稀释70倍),再依次加0.2mmol/L的硼酸缓冲液(pH9.0)1.0mL,0.5mL 6.0%重蒸苯酚和5.0mL 10%次氯酸钠,沸水浴加热10min后,迅速冰浴20min,待溶液出现蓝绿色后,加入60%(v/v)乙醇溶液4.0mL在640nm测定吸光值,绘制出标准曲线来计算GABA的含量.结果表明,该方法简便实用,相对误差小,适合实验室样品的快速检测.Based on the Berthelot color reaction,the orthogonal method was used to determinate the content of γ-aminobutyric acid produced form recombinant E.coli Glutamate decarboxylase transformation of L-Glutamate.The optimum conditions were determined as follows：2.0 mL 0.2 mol/L pH 6.0 acetate buffer（containing 0.1 mmol/L of PLP,0.2 mol/L of L-Glutamate,diluted 70 times）,incremented by 0.2 mmol/L of borate buffer（pH 9.0） 1.0 mL,0.5 mL 6.0% distilled phenol and 5.0 mL 10% sodium hypochlorite,boiling water bath heating for 10 minutes,being put in ice water immediately for 20 minutes,waiting for the solution to appear blue-green,adding to 60%（v/v） ethanol solution by 4.0 mL,determining at 640 nm and drawing a standard curve to calculate the content of GABA.The results showed that the method was simple and practical,the relative error was very small and suitable for rapid detection of laboratory samples.

关 键 词：Γ-氨基丁酸 谷氨酸脱羧酶 大肠杆菌工程菌 比色法 

分 类 号：Q93-33[生物学—微生物学] Q939.97