title: Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf.
abstract: An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-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 native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol(-1). It showed higher specificity with catechol (K(m) = 8 mM) as compared to 4-methylcatechol (K(m) = 10 mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.
authors: Department of Biotechnology, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, India.
authors: Sanjeev Kumar
authors: SK
authors: Diwakar
authors: Sarad Kumar
authors: SK
authors: Mishra
mesh_terms: D004156:Catechol Oxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D028521:Musa / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D028561:Transition Elements / Q000378:metabolism
chemical_list: D002396:Catechols; D004791:Enzyme Inhibitors; D062385:Hydroxybenzoates; D010940:Plant Proteins; D028561:Transition Elements; C009091:protocatechuic acid; D004156:Catechol Oxidase