Purification and Characterization of Ionically Unbound Polyphenol oxidase from Cinnamomum tamala syn. Cinnamomum Leaves
Author(s):
Abstract:
Polyphenol oxidase (EC. 1.10.3.1 PPO) an ionically unbound and thermostable enzyme was extracted from the leaves of Cinnamomum 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 (t1/2) 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 (Km=6.8mM). Among metal ions and reagents tested, Cu2+ indicating its role as cofactors, Fe2+, Hg2+, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K+, Mg2+, Co2+, kojic acid, L-ascorbic acid, ethylenediamine tetraacetic acid (EDTA), urea, sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.
Keywords:
Language:
English
Published:
Applied Food Biotechnology, Volume:2 Issue: 2, Spring 2015
Pages:
31 to 38
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