A METHOD FOR IMMOBILIZATION OF LIPOXYGENASE

A commercial preparation of soybean [Glycine max (L.) Merr.] lipoxygen ase (EC 1.13.11.12), an enzyme that catalyses the formation of fatty a cid hydroperoxides, was covalently immobilized on a commercially avail able carbonyldi-imidazole activated support. The degree of protein loa ding on to the support and the subsequent activity of immobilized lipo xygenase were found to be independent of the pH at which coupling was performed. Dialysis of lipoxygenase prior to immobilization did not en hance the coupling yield. As protein bound to the support increased, t he specific activity of lipoxygenase decreased. The reusability of imm obilized lipoxygenase was tested in tin aqueous buffer and in an octan e/aqueous buffer medium using linoleic acid as the substrate. In aqueo us buffer the immobilized preparation retained its activity even after seven cycles, whereas in the octane/buffer medium the activity of the immobilized preparation decreased to 60% of its original activity aft er seven,cycles. The optimal temperature for hydroperoxide formation w as 15 degrees C, with hydroperoxide yields decreasing at higher temper atures. Storage of immobilized lipoxygenase at 5 degrees C resulted in a loss from the support of approx. 5% of the protein in 25 days, but none thereafter. Lipoxygenase activity was stable at 5 degrees C, decr easing by only 5% in 6 months. The stability of immobilized lipoxygena se at 15 degrees C in aqueous buffer was approx. 10-fold greater than that of unbound lipoxygenase. The results show that immobilized lipoxy genase can be used in aqueous media as well as those containing organi c solvent.