Purification and characterization of Ocimum basilicum L. polyphenol oxidase.
A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be approximately 54 kDa. The contents of total phenolic and protein of O. basilicum L. extracts were determined. The total phenolic content of O. basilicum L. was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg 100 g(-1) on a fresh weight basis. The protein content was determined according to the Bradford method. The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the highest V(max)/K(m) value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for 4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature of 20, 40, and 50 degrees C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that optimum temperature and pH were dependent on the substrates studied. The enzyme activity with increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates decreased due to heat denaturation of the enzyme.
Dogan
Serap
S
Departments of Biology, Faculty of Science and Literature, University of Balikesir, 10100 Balikesir, Turkey. sdogan@balikesir.edu.tr
Turan
Pinar
P
Dogan
Mehmet
M
Arslan
Oktay
O
Alkan
Mahir
M
D004156:Catechol Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D018649:Ocimum basilicum / Q000201:enzymology*; D010940:Plant Proteins / Q000032:analysis; D013379:Substrate Specificity; D013696:Temperature
D010940:Plant Proteins; D004156:Catechol Oxidase