A New Method for Determining Activity of CMEPA Hydrolase

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 10-50 μmol·L^-1. The linear equation goes as y=0.014 6x-0.005 4, R²=0.9988. 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-aminoantipyrene colorimetric method does not vary much in determining enzymatic activity of T3-6 crude enzyme. This method is also applicable to the determination of other derivatives of aniline and phenol.