Purification and properties of diethyl p-phthalate esterase from Ochrobactrum anthoropi 6-2b

Abstract

An esterase which hydrolyzes diethyl p-phthalate (diethyl terephthalate, DET) was obtained from Ochrobactrum anthoropi 6-2b. It was purified 19.1 fold to electrophoretic homogeneity with a yield of 26%. The purified esterase had a specific activity of 32.2 μmol -min-1 · mg-1-protein based on the hydrolysis of DET at pH 7.0 and 30°C. The enzyme showed maximum activity at pH 8.0 and was stable at pH 6.0-8.0 with optimum stability at pH 6.0. Its optimal temperature in reaction was 50°C, and it was stable below 40°C. Esterase activity was 89% inhibited by adding 1.0 mM Ni2+ and 82% inhibited by 1.0 mM phenylmethylsulfonyl fluoride. The esterase obeyed Michaelis-Menten kinetics, and in the hydrolysis of DET, Vmax and Km were 37.3 μmol · min-1 · mg-1-protein and 1.17 mM, respectively. It was able to hydrolyze a number of p-phthalates and o-phthalates. Its molecular weight was 40,000 by gel filtration chromatography and 20,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.