Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe

The potential application of lipoxygenase as a versatile biocatalyst in enz yme technology is limited by its poor stability. Two types of soybean lipox ygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two st ep anion exchange chromatography. Four different commercially available sup ports: CNBr Sepharose 4B, Fractogel (R) EMD Azlactone, Fractogel (R) EMD Ep oxy, and Eupergic (R) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activi ty and good stability after immobilization on CNBr Sepharose 4B and Fractog el (R) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within t he first 5 days after immobilization on epoxy activated supports (Eupergit (R) C and Fractogel (R) EMD Epoxy) in high ionic strength buffers. Stabiliz ation of the biocatalyst on these supports was achieved by careful adjustme nt of the immobilization conditions. When immobilized in phosphate buffer o f pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobil ized enzyme increased 20 fold. The dependence of the stability of LOX immob ilized on epoxy activated supports on the coupling conditions was attribute d to a modulation of the ligand environment of the iron in the active site and consequently its reactivity. (C) 2001 Elsevier Science Inc. All rights reserved.