Partial purification and some properties of polyphenol oxidase from Laurus nobilis L.
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2006
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Abstract
Polyphenol oxidase from laurel leaves tissue (Laurus nobilis L.) was isolated by (NH4)2SO4 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°C for catechol, 35°C for 4-methylcatechol, 30°C for pyrogallol and gallic acid, 25°C for DL-dopa and L-dopa. Half lives of PPO activity were 28 min. at 60°C and 7 min. at 70°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 (KM and Vmax 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/ml for pyrogallol, 66.7 mM and 7142 U/ml for L-dopa, 70.9 mM and 5000 U/ml for DL-dopa). L-tyrosine was also tested but was not oxidized by Laurus nobilis PPO. The I50 value was found to be 7.60×10-6 M for dithiothreitol, 1.60×10-5 M for β-mercaptoethanol, 2.40×10-5 M for glutathione, 3.00×10-5 M for sodium thiosulphate, 0.76×10-4 M for sodium azide, 0.80×10-4 M for thiourea, 0.84×10-4 M for sodium metabisulfite, 1.45×10 -4 M for L-csyteine, 5.00 ×10-4 for ascorbic acid, 6,00×10-4 M for oxalic acid, 1.11×10-2 M for citric acid and 4.40×10-2 M for EDTA. Various amino acids such as L-csyteine, 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.