Partial purification and some properties of polyphenol oxidase from Laurus nobilis L.
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Polyphenol oxidase from laurel leaves tissue (Laurus nobilis L.) was isolated by (NH4)(2)SO4 precipitation and dialysis. Stable and highly active PPO extracts were obtained using 1.5% (w/v) Triton X-100 and 1.0% (w/v) polyethylene glycol (PEG) in 0.05 M potassium phosphate buffer pH 7.0. The optimum pH values were found to be 7.0 for catechol, DL-dopa and L-dopa, 6.5 for 4-methylcatechol, 6.0 for pyrogallol and 5.0 for gallic acid. Laurus nobilis PPO is more stable at basic pH than acidic pH values. The optimum temperature was found to be 40 degrees C for catechol, 35 degrees C for 4-methylcatechol, 30 degrees C for pyrogallol and gallic acid, 25 degrees C for DL-dopa and L-dopa. Half lives of PPO activity were 28 min. at 60 degrees C and 7 min. at 70 degrees C with catechol. Ea value was calculated from the Arhenius equation 16.628 kj/mol for catechol as Substrate. Polyphenol oxidase showed activity toward catechol, 4-methylcatechol, gallic acid, pyrogallol, L-dopa and DL-dopa (K,, and V,, values were 8.3 mM and 15538 U/ml for catechol. 14.8 mM and 11300 U/ml for 4-methylcatechol, 15.6 mM and 9153 U/ml for gallic acid, 28.0 mM and 8835 U/mI for pyrogallol, 66.7 mM and 7142 U/ml for L-dopa, 70.9 mM and 5000 U/mI for DL-dopa). L-tyrosine was also tested but was not oxidized by Laurus nobilis PPO. The 1511 Value was found to be 7.60x 10(-6) M for dithiothreitol, 1.60 x 10(-5) M for beta-mercaptoethanol. 2.40 x 10(-5) M for glutathione, 3.00 x 10(-5) M for sodium thiosulphate, 0.76x10(-4) M for sodium azide, 0.80x10(-4) M for thiourea, 0.84x10(-4) M for sodium metabisulfite, 1.45x10(-4) M for L-csyteine, 5.00 x 10(-4) for ascorbic acid, 6,00x10(-4) M for oxalic acid, 1.1 x 10(-2) M for citric acid and 4.40x 10(-2) M for EDTA. Various amino acids such as L-csyteme, L-glycine, L-arginine, L-phenyl alanin, L-glutamic acid and L-aspartic acid have been investigated and the results showed that L-cysteine was the most effective inhibitors. NaCl, CoCl2, CaCl2, were poor inhibitors of enzyme at 1 mM. Fe++, Mg++, Mn++, Zn++ ions did not show away significant effect on partially purified enzyme activity.