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Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040

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Abstract

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s−1 were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 31200050 and 21276082) and Ministry of Science and Technology, People’s Republic of China (Nos. 2011AA02A210 and 2011CB710800).

Ethical Statement

This manuscript is original and has not been published and will not be submitted elsewhere for publication. No data has been fabricated or manipulated (including images) to support our conclusions. No data, text, or theories by others are presented as if they were our own.

The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results.

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The authors declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human participants or animals performed by any of the authors.

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Authors and Affiliations

  1. Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Jin-Gang Yin, Guo-Chao Xu, Gao-Wei Zheng & Jian-He Xu

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  1. Jin-Gang Yin
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  2. Guo-Chao Xu
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  3. Gao-Wei Zheng
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Correspondence to Gao-Wei Zheng or Jian-He Xu.

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Fig. S1

Effect of pH on AbMBH activity. The relative activity was determined using dl-menthyl benzoate as the substrate. Enzyme assay was performed in different buffers (100 mM) within a pH range of 6.0–10.0 (potassium phosphate, 6.0–8.5; Tris–HCl, 8.0–9.0; Na2CO3–NaHCO3, 9.0–10.0) at 30°C. Relative activity was expressed as a percentage of maximum activity under the experimental conditions. (DOC 42 kb)

Fig. S2

Effect of temperature on AbMBH activity. Relative activity was expressed as a percentage of maximum activity under the experimental conditions. (DOC 42 kb)

Fig. S3

Effect of temperature on AbMBH stability. Diamond, 30 °C (inserted graph); circle, 40 ºC; and square, 50 °C. Samples were withdrawn at different time intervals to estimate the residual activity using the standard assay. Residual activity was expressed as a percentage of the activity measured initially without any pre-incubation. (DOC 36 kb)

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Yin, JG., Xu, GC., Zheng, GW. et al. Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040. Appl Biochem Biotechnol 176, 1102–1113 (2015). https://doi.org/10.1007/s12010-015-1632-0

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