Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid.
Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9- and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30-45 and 45-80% saturated (NH4)2SO4 precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH4)2SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorella is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.
Nuñez
Alberto
A
USDA, ARS, ERRC, Fats, Oils and Animal Coproducts Unit, Wyndmoor, Pennsylvania 19038, USA. anunez@arserrc.gov
Savary
Brett J
BJ
Foglia
Thomas A
TA
Piazza
George J
GJ
D002708:Chlorella / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D019787:Linoleic Acid / Q000378:metabolism*; D008041:Linoleic Acids / Q000737:chemistry / Q000378:metabolism; D008054:Lipid Peroxides / Q000737:chemistry / Q000378:metabolism*; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D013054:Spectrophotometry, Atomic
D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C049606:9-hydroperoxy-11,12-octadecadienoic acid; D019787:Linoleic Acid; D006861:Hydrogen Peroxide; D008084:Lipoxygenase