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Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

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An Erratum to this article was published on 25 April 2014

Abstract

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4∼20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K M was 210 μM and k cat was 3.4 s−1. Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.

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Acknowledgments

The authors thank Dr. Mal-Gi Choi for CD analysis. This work was supported by the Daegu University Research Grant, 2010 (to S.-H.J.).

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Authors and Affiliations

  1. Department of Biomedical Science, Daegu University, Gyeongsan, 712-714, South Korea

    ChangWoo Lee, Junyoung Kim, Seunghee Hong, Bonlon Goo & Sei-Heon Jang

  2. Gyeongsan Science High School, Gyeongsan, 712-260, South Korea

    Seungyeon Lee

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  1. ChangWoo Lee
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  2. Junyoung Kim
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  3. Seunghee Hong
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Correspondence to Sei-Heon Jang.

Additional information

An erratum to this article is available at http://dx.doi.org/10.1007/s12010-014-0919-x.

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Lee, C., Kim, J., Hong, S. et al. Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii . Appl Biochem Biotechnol 169, 29–40 (2013). https://doi.org/10.1007/s12010-012-9947-6

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