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 30-43 and 45-80% saturated (NH4)(2)SO4 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)(2)SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SIDS-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.