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Recombinant Maize 9-Lipoxygenase: Expression, Purification, and Properties

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

Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, ul. Lobachevskogo 2/31, 420111 Kazan, Russia; fax: (843) 292-7347; E-mail: tarasova@mail.knc.ru

* To whom correspondence should be addressed.

Received July 17, 2009; Revision received November 16, 2009
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.
KEY WORDS: maize 9-lipoxygenase, expression of 9-lipoxygenase, chromatography of 9-lipoxygenase, GC-MS analysis, HPLC

DOI: 10.1134/S0006297910070072