Purification and characterization of lipoxygenase from the thermophilic fungus Thermomyces lanuginosus

A thermostable intracellular lipoxygenase from Thermomyces lanuginosus was purified to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by ion-exchange chromatography on diehylaminoethyl (DEAE)-Sepharose, ion-exchange chromatography on carboxymethyl (CM)-Sepharose and Phenyl-Sepharose hydrophobic interaction chromatography. The molecular weight of the enzyme was estimated to be 100 kDa by SDS-PAGE. The lipoxygenase exhibited maximal activities at pH 6.0. The optimum temperature for the activity was 55 degreesC. The enzyme was thermostable at 50 degrees with half- lives at 60 degrees of 20 min and 70 degrees of about 7 min. The lipoxygenase activity was completely lost by heating at 80 degrees for 5 min. The lipoxygenase catalyzed the oxidation of linoleic acid into 13-L-hydroperoxy-9,11(Z,E)-octadecadienoic acid(13-HPOD) as the major product. The enzyme had an apparent Km of 8.5 muM and V-max of 10.8 mu mol mg(-1) min(-1) with linoleic acid as substrate.