Novel peroxidase from bitter leaf (Vernonia amygdalina): Purification, biochemical characterization and biotechnological applications

Bitter leaf could serve as a cost-effective and alternative source of plant peroxidase for several applications. In this study, the bioavailability, biochemical properties and potentials of bitter leaf peroxidase in some biotechnological applications are described. The enzyme was first purified using a fast and cost-effective approach. The combination of aqueous two-phase partitioning and gel filtration chromatography on Bio Gel P-100 was very efficient resulting to a percentage yield and final purification fold of 76% and 7.1 respectively. Native and subunit molecular weights of the purified peroxidase were 41.2 +/- 2.1 kDa and 39.5 +/- 1.0 kDa, respectively, revealing the monomeric nature of bitter leaf POD. The optimum pH was 6.0 while that of temperature was 30 degrees C. The estimated kinetic constants Km were 2.5 +/- 0.1 mM and 5.1 +/- 0.2 mM for odianisidine and hydrogen peroxide respectively while maximum velocity (Vmax) was 3010.6 +/- 21 units/mg protein. The enzyme was activated in the presence of organic solvents (at up to 30% chloroform). It also retained stability (>= 60%) at up to 50% concentration of acetone, methanol and ether. Chlorides of Ca2+, Na+ and Cu2+ activated the enzyme whereas Al3+ and Fe3+ had inhibitory effects. Bitter leaf POD was covalently bonded with casein to form higher molecular adduct, establishing its potentials as a reporter enzyme. In conclusion, bitter leaf peroxidase possesses remarkable biochemical properties that could be of interest in several industrial and biotechnological applications.

Automated summary

Bitter leaf peroxidase is a cost-effective and alternative source of plant peroxidase with potential applications in various biotechnological fields. The enzyme was purified using a fast and cost-effective method and showed high efficiency in terms of yield and purification fold. It exhibited optimal pH and temperature conditions, and its kinetic constants were determined. The enzyme showed activation in the presence of certain organic solvents and retained stability at high concentrations of acetone, methanol, and ether. It also displayed selective activation and inhibitory effects in the presence of certain metal chlorides. Covalent bonding of bitter leaf peroxidase with casein established its potential as a reporter enzyme.