Abstract:
In this study, Polyphenol oxidase from scent leaf was isolated and partially purified by a procedure that involved ammonium sulphate precipitation, ion exchange chromatography on DEAE Sephacel and gel filtration on Sephadex G-100. Some physicochemical properties of the partially purified enzyme such as the effects of pH and temperature on the activity and stability, pH dependence activation by Sodium dodecyl sulphate, effects of metal ions and inhibitors, substrate specificity, and kinetic parameters were investigated. The purification fold and recovery achieved for the partially purified polyphenol oxidase were 6.87 and 0.5%. The enzyme was optimally active at 50ºC and had two optima pH of 5.0 and 7.0. There was considerable activation of the enzyme with Sodium dodecyl sulphate between pH 5.0 - 7.0 with one optimum at pH 7.0. The enzyme was relatively stable at 40ºC and 50ºC while at pH 5.0, 6.0, and 7.0, the enzyme maintain above 70% relative activity of the initial a 2 hr incubation. A complete inhibition was demonstrated by Fe2+ and Hg2+. Zn2+, Mg2+, and Ca2+ partially inhibited the enzyme while Cu2+ markedly activated scent leaf Polyphenol oxidase. The enzyme kinetics has a Km of 3.2 mM, 5.29 mM and 83.33 mM for Catechol, L-dihydroxyl phenol alanine, and tyrosine respectively. The Vmax was 45.46 unit/min, 28.57 unit/min and 20.08 unit/min for Catechol, L- L-dihydroxyl phenol alanine, and Tyrosine respectively. The Polyphenol oxidase from scent leaf was shown to be specific to o-dihydroxy phenol. In conclusion, the result suggested Polyphenol oxidase will not only contribute to the brownish/darkish colouration of the liquors of Ocimum gratissimum but can be properly managed under some certain physicochemical properties to minimize the brownish/darkish presentations of scent leaf liquors.
