Purification and characterization of soluble Cichorium intybusVar. silvestre lipoxygenase.
A water-soluble lipoxygenase enzyme (EC 1.13.11.12; LOX) occurring in the red cultivar produced in the geographical area of Chioggia (Italy) of Cichorium intybus var. silvestre was isolated and characterized. The molecular mass of the enzyme was estimated to be 74,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The isoelectric point was pH 6.85. The optimum values of pH, ionic strength, and temperature, shown by isoresponse surface calculated by a randomized multilevel factorial design, were 7.58, 30 mM, and 38.5 degrees C, respectively. The enzyme showed high specificity toward linoleic acid, and the study of the variation of linoleic acid concentration between 30 and 300 microM, in the presence of Tween 20 at a concentration lower than the critical micelle concentration (0.01 v/v), resulted in a typical Michaelis-Mentem curve with KM and Vmax values of 1.49 x 10(-4) M and 2.049 microM min(-1) mg(-1), respectively. The biochemical properties, the kinetic parameters found, and the carotene-bleaching activity shown in aerobic conditions seem to indicate that the isolated enzyme is a lipoxygenase type III according to the indications given for soybean isoenzymes.
Daglia
Maria
M
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy.
Aceti
Camilla
C
Giorgetti
Sofia
S
Papetti
Adele
A
Gazzani
Gabriella
G
D018651:Cichorium intybus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D009994:Osmolar Concentration; D013696:Temperature
D008084:Lipoxygenase