Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13-hydroperoxides of linoleic and linolenic acids
An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13-hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo-product, 13-oxo-9(Z),11(E)tridecadienoic 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 derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13-hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13-OTA along with the 13-ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13-and 9-hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH 4 ) 2 SO 4 precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.