Abstract
The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel® EMD Azlactone, Fractogel® EMD Epoxy, and Eupergit® C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel® EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit® C and Fractogel® EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.
| Original language | English |
|---|---|
| Journal | Enzyme and Microbial Technology |
| Volume | 28 |
| Issue number | 2-3 |
| Pages (from-to) | 168-175 |
| Number of pages | 8 |
| ISSN | 0141-0229 |
| DOIs | |
| Publication status | Published - 01.02.2001 |
Funding
We thank Prof. Dr. W. Calmano and Mrs. Irene Brauer (Technical University of Hamburg-Harburg, Germany) for kindly running the AAS measurements. We are also grateful to Prof. Dr. A.X Trautwein (Medical University of Lübeck, Germany) for providing us the facilities to carry out the EPR measurements. This work was supported by the German Academic Exchange Service (DAAD) and German Research Foundation (DFG, Graduiertenkolleg/Biotechnologie, GK-95/3-97).
