Role of calcium in enhancing the activity and thermal stability of a new cationic peroxidase purified from euphorbia tirucalli latex

Abstract EN

A new cationic peroxidase from Euphorbia tirucalli (pencil cactus) latex was purified to homogeneity using benzene fractionation, gel filtration and cation-exchange chromatography.

The purified enzyme was found to be monomeric with a molecular weight of 44 k Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The purified enzyme had a broad specificity towards some phenolic substrates in the order of 2, 2- azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) > guaiacol o-phenylenediamine > 4-aminoantipyrene, whereas no affinity towards ascorbic acid and o-dianisidine was recorded.

The enzyme had pH and temperature optima at 7.0 and 40°C, respectively.

Study of kinetic parameters demonstrated that ABTS had the highest affinity towards ELP, where Km, V max and V max / Km values were 0.503 mM, 500 U / assay and 994.04 U/mM, respectively.

ELP was stable from 10˚C up to 60°C and lost about 70 % of its activity at 70°C.

The thermal inactivation profile of ELP in absence of Ca2+ is biphasic and characterized by a rapid decline in activity on exposure to heat, followed by a more gradual decrease in activity on continued exposure.

However, the purified enzyme exhibited increased thermal stability in the presence of calcium ions.

Furthermore, the activity of purified enzyme was enhanced by ~ 550 % in the presence of 15 mM CaCl2, suggesting a pivotal role for Ca2+ in conferring structural stability to the home environment and in retaining the active site of ELP.

Most of the examined metal ions (except for Ca2+ and Mg2+) and compounds had differential inhibitory effects on ELP activity.

In conclusion, a locally available plant (Euphorbia tirucalli) could be a potential candidate source for peroxidase, the most widely used enzyme in industrial and biomedical applications.

In addition, calcium was found to be essential for enhancing enzymatic activity and thermal stability of the purified Euphorbia tirucalli latex peroxidase.