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 H₂O₂ were 8 mM and 1.8 mM, 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.

Keywords:

• Citrus medica
• inactivation kinetics
• purification
• soluble peroxidase
• thermostable

ACKNOWLEDGMENT

The authors are thankful to the Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur, for kind support.