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

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Abstract

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 pNPB as the substrate. The Km, Vmax, kcat and kcat/Km 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 pNPB 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.

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Acknowledgements

This work was supported by Zhejiang Provincial Natural Science Foundation (LY18B020021) and (LY18E090010).

Supporting information

Supplementary Fig. S1—The amino acid sequences (red) of the peptide fragment (from the esterase BaCE) were found to match the carboxylesterase from Bacillus megaterium

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Yin-Jun Zhang, Chang-Sheng Chen, Hao-Tian Liu, Jia-Lin Chen, Ying Xia & Shi-Jin Wu

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  1. Yin-Jun Zhang
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  2. Chang-Sheng Chen
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  3. Hao-Tian Liu
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Correspondence to Yin-Jun Zhang or Shi-Jin Wu.

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Zhang, YJ., Chen, CS., Liu, HT. et al. Purification, identification and characterization of an esterase with high enantioselectivity to (S)-ethyl indoline-2-carboxylate. Biotechnol Lett 41, 1223–1232 (2019). https://doi.org/10.1007/s10529-019-02727-w

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