PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE SOLUBLE PEROXIDASE FROM Citrus medica LEAF

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 32kD as determined by SDS-PAGE. The enzyme was optimally active at pH 6.0 and 50 degrees C temperature. The enzyme was active in wide range of pH (5.08.0) and temperature (3080 degrees C). From the thermal inactivation studies in the range of 6075 degrees C, the half-life (t1/2) values of the enzyme ranged from 8 to 173min. The inactivation energy (Ea) value of POD was estimated to be 21.7kcal mol1. The Km values for guaiacol and H2O2 were 8mM and 1.8mM, respectively. This enzyme was activated by some metals and reagents such as Ca2+, Cu2+, Mg2+, Co2+, ferulic acid, and indole acetic acid (IAA), while it was inhibited by Fe2+, Zn2+, Hg2+, and Mn2+, L-cysteine, L-proline, and protocatechuic acid.