PARTIAL-PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE IN CHLORELLA-PYRENOIDOSA

Lipoxygenase was partially purified, from a crude homogenate of Chlorella pyrenoidosa, by solid (NH4)(2)SO4 at 40-80% satn. The partially purified enzyme exhibited an optimum pH activity at 4.5; however, there was a dramatic decrease in lipoxygenase activity when the pH of the enzymic reaction was increased. The apparent K-m and V-max. values, obtained by Lineweaver-Burk plots, were determined to be 9.12 x 10(-5) M and 0.401 mu mol of linoleic acid/min per mg of protein respectively. KCN markedly inhibited lipoxygenase activity by 58.2% at 0.5 mM it was an uncompetitive inhibitor, as indicated by the corresponding K-m and V-max. values of 4.75 x 10(-5) M and 0.246 mu mol of linoleic acid/min per mg of protein. The addition of 5 mM sodium EDTA to the reaction medium produced a noticeable 8-fold increase in enzymic activity. Substrate specificity was investigated using free linoleic and linolenic acids, as well as mono-, di- and tri-linoleoylglycerol. The partially purified enzyme demonstrated a preference for free linoleic acid in comparison with free linolenic acid and its esters, mono-, di- and tri-linoleoylglycerol. Native PAGE indicated the presence of five bands of molecular mass varying between 67 and 140 kDa.