title: Purification and characterization of a thermostable soluble peroxidase from Citrus medica leaf.
abstract: A soluble and thermostable peroxidase enzyme (POD) was extracted from the leaf of Citrus medica. The enzyme was purified 15.10-fold with a total yield of 28.6% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme came as a single band on native polyacrylamide gel electrophoresis (PAGE) as well as sodium dodecyl sulfate (SDS) PAGE. The molecular mass of the enzyme was about 32 kD as determined by SDS-PAGE. The enzyme was optimally active at pH 6.0 and 50°C temperature. The enzyme was active in wide range of pH (5.0-8.0) and temperature (30-80°C). From the thermal inactivation studies in the range of 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 8 to 173 min. The inactivation energy (Ea) value of POD was estimated to be 21.7 kcal mol(-1). The Km values for guaiacol and H(2)O(2) were 8 mM and 1.8 mM, respectively. This enzyme was activated by some metals and reagents such as Ca(2+), Cu(2+), Mg(2+), Co(2+), ferulic acid, and indole acetic acid (IAA), while it was inhibited by Fe(2+), Zn(2+), Hg(2+), and Mn(2+), L-cysteine, L-proline, and protocatechuic acid.
authors.affiliation: Department of Biotechnology, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, India.
authors.forename: Ruckminee
authors.initials: R
authors.lastname: Mall
authors.forename: Gaurav
authors.initials: G
authors.lastname: Naik
authors.forename: Usha
authors.initials: U
authors.lastname: Mina
authors.forename: Sarad Kumar
authors.initials: SK
authors.lastname: Mishra
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D002850:Chromatography, Gel; D002957:Citrus / Q000201:enzymology*; D003300:Copper / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D006139:Guaiacol / Q000737:chemistry; D006207:Half-Life; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification*; D018515:Plant Leaves / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007477:Ions; D010940:Plant Proteins; D006139:Guaiacol; D003300:Copper; D006861:Hydrogen Peroxide; D010544:Peroxidases; D008274:Magnesium; D000645:Ammonium Sulfate