Recombinant maize 9-lipoxygenase: expression, purification, and properties.
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 degrees 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, beta-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.
Osipova
E V
EV
Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, Russia.
Chechetkin
I R
IR
Gogolev
Y V
YV
Tarasova
N B
NB
D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D010940:Plant Proteins; D011994:Recombinant Proteins; D008084:Lipoxygenase