Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into ( S )-( E )-10-hydroxy-8-octadecenoic acid.
The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.
Busquets
M
M
Departamento de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franqués 1, Barcelona 08028, Spain.
Deroncelé
V
V
Vidal-Mas
J
J
Rodríguez
E
E
Guerrero
A
A
Manresa
A
A
D001711:Biotransformation; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism*; D009829:Oleic Acids / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013816:Thermodynamics
C107324:10-hydroxy-8-octadecenoic acid; D009829:Oleic Acids; D019301:Oleic Acid; D008084:Lipoxygenase