一种检测2′－甲基－6′－乙基－2－氯乙酰苯胺水解酶活性的方法

摘要

建立了一种快速测定Delftia acidovorans T3－6菌株2′－甲基－6′－乙基－2－氯乙酰苯胺（ CMEPA）水解酶活性的方法。在20 mmol·L－1 Tris－HCl（ pH 值为7�0）缓冲液中，10～50μmol·L－1的CMEPA水解产物2－甲基－6－乙基苯胺（MEA）在铁氰化钾催化下与4－氨基安替比林反应生成紫红色物质，该物质在535 nm处的吸光度（y）与MEA浓度（ x）呈正相关（ y＝0�0146x－0�0054，R2＝0�9988）。底物CMEPA、十二烷基硫酸钠（ SDS）、甲醇等有机试剂以及1 mmol·L－1金属离子对显色反应无显著影响，温度和pH值对显色反应影响显著。4－氨基安替比林显色法对菌株T3－6粗酶液酶活性的测定结果与二氯甲烷萃取－紫外分光光度法无显著差异。该方法也适用于其他苯胺苯酚类衍生物的测定。%A new method for rapid determination of the activity of 2′-methyl-6′-ethyl-2-chloroacetanilide(CMEPA) hydrolase in Delftia acidovorans T3-6 was established. CMEPA hydrolase can hydrolyze CMEPA into 2-methyl-6-ethyl-aniline ( MEA) , which, in turn, reacts with 4-aminoantipyrene in 20 mmol·L-1 Tris⁃HCl ( pH 7�0) buffer to produce a compound, purple in color, with absorbancy at 535 nm( y) positively related to the concentration of MEA( x) in the range of 10-50μmol·L-1 . The linear equation goes as y=0�014 6x-0�005 4, R2=0�998 8. The compounds of CMEPA, SDS, organic reagent such as methanol and 1 mmol·L-1 metal ions have no significant effect on chromogenic reaction, whereas temperature and pH does. Compared with the dichloromethane extraction⁃UV spectrophotometry method, the 4-aminoanti⁃pyrene colorimetric method does not vary much in determining enzymatic activity of T3-6 crude enzyme. This method is al⁃so applicable to the determination of other derivatives of aniline and phenol.