Carboxylesterase 3 (CES3) also known as Liver carboxylesterase 3 is a serine esterase and a member of a large family of carboxylesterases. The protein Involved in the detoxification of xenobiotics and in the activation of ester and amide prodrugs. Hydrolyzes aromatic and aliphatic esters, but has no catalytic activity toward amides or a fatty acyl-CoA ester. Hydrolyzes the methyl ester group of cocaine to form benzoylecgonine. Thus, the recombinant mouse CES3 activity was measured by its ability to hydrolyze 4-Nitrophenyl acetate (4-NPA) to 4-Nitrophenol. The reaction was performed in 50 mM Tris, pH 7.5( Assay Buffer), initiated by addition 50 μL of various concentrations of CES3 (dilute by Assay Buffer) to 50 μL of 2 mM Substrate 4-NPA(100 mM stock in Acetone, dilute by deionized water). Incubated at 37℃ for 10min, then read at a wavelength of 400 nm.

One unit of enzyme activity is defined as the 1μg of enzyme required to convert 1pmol of 4-Nitrophenyl acetate to 4-Nitrophenol in 1min at 37°C. The specific activity of recombinant mouse CES3 is 900 pmol/min/μg.