Purification and characterization of ionically unbound polyphenol oxidase from cinnamomum tamala syn. cinnamomum leaves

ABSTRACT

Polyphenol oxidase [EC. 1.10.3.1 PPO] an ionically unbound and thermostable enzyme was extracted from the leaves ofCinnamomum tamala. The enzyme was purified 2.63-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate [SDS] PAGE. It was found to be monomeric protein with molecular mass of about 25 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 25 kD. The enzyme was optimally active at pH 7.0 and 50oC temperature. The enzyme was active in wide range of pH [4.0-9.0] and temperature [30-90oC]. From the thermal inactivation studies in the range 60-80oC, the half-life [t[1/2]] values of the enzyme ranged from 19 to 72 min. The inactivation energy [Ea] value of PPO was estimated to be 94.5 kJ mol[-1] . It showed higher specificity with substrate catechol [K[m]=6.8mM]. Among metal ions and reagents tested, Cu[+2] indicating its role as cofactors, Fe[+2], Hg[+2], protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K[+], Mg[+2], Co[+2], kojic acid, L-ascorbic acid, ethylenediamine tetraacetic acid [EDTA], urea, sodium azide, beta-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme