Purification, identification and characterization of an esterase with high enantioselectivity to ( S )-ethyl indoline-2-carboxylate

Objective To purify an esterase which can selectively hydrolyze ( R,S )-ethyl indoline-2-carboxylate to produce ( S )-indoline-2-carboxylic acid and characterize its enzymatic properties. Results An intracellular esterase from Bacillus aryabhattai B8W22 was isolated and the purified protein was identified as a carboxylesterase by MALDI-TOF mass spectrometry. The enzyme (named BaCE) was 59.03-fold purification determined to be of approximately 35 kDa. Its specific activity was 0.574 U/mL with 20% yield. The enzyme showed maximum activity at pH 8.5 and 30 °C and was stable at 20–30 °C using p NPB as the substrate. The K m , V max , k cat and k cat /K m of the esterase were 0.52 mM, 6.39 μM/min, 26.87 min −1 and 51.67 mM/min, respectively. The esterase demonstrated high enantioselectivity toward ( S )-ethyl indoline-2-carboxylate with 96.55% e.e. p at 44.39% conversion, corresponding to an E value of 133.45. Conclusions In this study, a new esterase BaCE with an apparent molecular mass of 35 kDa was purified to homogeneity for the first time. The esterase from Bacillus aryabhattai B8W22 was isolated with a purification more than 59-fold and a yield of 20% by anion exchange chromatography and hydrophobic interaction chromatography. And its biochemical characterization were described in detail with p NPB as substrate. It displayed high enantioselectivity toward ( S )-ethyl indoline-2-carboxylate. We next plan to highly express esterase BaCE in Escherichia coli , and apply it to industrial production of ( S )-indoline-2-carboxylic acid.