Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible forthe cleavage of the 13-hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a h ydroperoxide lyase (HPLS), cleaves the 13-hydroperoxide derivatives of lino leic and linolenic acids into a volatile C5 fragment and a C13 ore-product, 13-oxo-9(Z), 11 (E)trideca-dienoic acid (13-OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13-hydroperoxide deriva tive of linoleic acid, whereas a more complex mixture of hydrocarbons was f ormed when the 13-hydroperoxide derivative of linolenic acid was the substr ate. Analysis of the nonvolatile products by GC/MS and liquid chromatograph y/mass spectrometry (LC/MS) indicated the formation of 13-OTA along with th e 13-ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincident al in purified fractions with lipoxygenase activity that produced the 13- a nd 9-hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a conseque nce of hydroperoxide lyase activity as previously thought, but was due to a naerobic lipoxygenase activity This enzyme fraction was purified by (NH4)(2 )SO4 precipitation, gel filtration, and hydrophobic interaction chromatogra phy. The purified enzyme has an approximate MW of 120 KDa and maximum activ ity at pH 8.0.