title: Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.
abstract: 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 k(cat)/K(m) value; a beta-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 beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.
authors.affiliation: Istituto di Biochimica delle Proteine ed Enzimologia, Consiglio Nazionale delle Ricerche, Napoli, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Barone
authors.forename: R
authors.initials: R
authors.lastname: Briante
authors.forename: S
authors.initials: S
authors.lastname: D'Auria
authors.forename: F
authors.initials: F
authors.lastname: Febbraio
authors.forename: C
authors.initials: C
authors.lastname: Vaccaro
authors.forename: L
authors.initials: L
authors.lastname: Del Giudice
authors.forename: G M
authors.initials: GM
authors.lastname: Borrelli
authors.forename: N
authors.initials: N
authors.lastname: Di Fonzo
authors.forename: R
authors.initials: R
authors.lastname: Nucci
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D005433:Flour; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D014908:Triticum / Q000201:enzymology*
chemical_list: D010446:Peptide Fragments; D008084:Lipoxygenase