Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 ± 5 kDa. Among the substrates tested, linoleic acid showed the highest kcat/Km value; a β-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6.8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on β-carotene, both assayed at 25 °C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 °C for linoleic acid and at 60 °C for bleaching activity on β-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 °C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of α- and β-structures. Near-UV circular ...