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Recombinant maize 9-lipoxygenase: Expression, purification, and properties

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Abstract

Expression of maize 9-lipoxygenase was performed and optimized in Escherichia coli Rosetta(DE3)pLysS. The purity of recombinant protein obtained during Q-Sepharose and Octyl-Sepharose chromatographies in an LP system at 4°C was >95%. Maximum activity of the lipoxygenase reaction was observed at pH 7.5. Enzyme stability was studied at pH 4.5 to 9.5 and in the presence of different compounds: phenylmethanesulfonyl fluoride, β-mercaptoethanol, ammonium sulfate, and glycerol. HPLC and GC-MS analysis showed that enzyme produced 99% 9S-hydroperoxide from linoleic acid. 13-Hydroperoxide (less than 1%) consisted of S- and R-enantiomers in ratio 2 : 3.

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Abbreviations

GC-MS:

gas chromatography-mass spectrometry

HPLC:

high performance liquid chromatography

IPTG:

isopropyl β-D-1-thiogalactopyranoside

LB:

Luria-Bertani (medium)

PMSF:

phenylmethanesulfonyl fluoride

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Authors and Affiliations

  1. Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, ul. Lobachevskogo 2/31, 420111, Kazan, Russia

    E. V. Osipova, I. R. Chechetkin, Y. V. Gogolev & N. B. Tarasova

Authors
  1. E. V. Osipova
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  2. I. R. Chechetkin
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  3. Y. V. Gogolev
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  4. N. B. Tarasova
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Corresponding author

Correspondence to N. B. Tarasova.

Additional information

Published in Russian in Biokhimiya, 2010, Vol. 75, No. 7, pp. 978–983.

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Osipova, E.V., Chechetkin, I.R., Gogolev, Y.V. et al. Recombinant maize 9-lipoxygenase: Expression, purification, and properties. Biochemistry Moscow 75, 861–865 (2010). https://doi.org/10.1134/S0006297910070072

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  • DOI: https://doi.org/10.1134/S0006297910070072

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