A novel acid phosphatase from cactus (Opuntia megacantha Salm-Dyck) cladodes: Purification and biochemical characterization of the enzyme

Abstract

Acid phosphatase (ACP) plays an important role in regulating phosphate nutrition in plants. Herein, for the first time, a novel ACP from Opuntia megacantha Salm-Dyck cladodes was purified to homogeneity and biochemically characterized. Specific activity of 8.78 U/mg was obtained with 11.29-fold purification and 15% yield. ACP was purified as monomer with molecular weight of 44 kDa as determined by SDS-PAGE under denaturing and nondenaturing conditions. Optimum pH and temperature for ACP activity was 5.5 and 60 °C, respectively. Thermodynamic parameters (E_a, ΔH, ΔG and ΔS) were also determined. ACP activity was stimulated by Ca²⁺, strongly inhibited by Cu²⁺ and Fe³⁺, and moderately inhibited by Mg²⁺ and Zn²⁺. Br^-, CN^-, F^-, I^- and N₃^- weakly inhibited ACP activity, where more than 70% of enzyme activity was remained at 5 mM. In addition, effect of β-ME, Cys, DTT, EDTA, H₂O₂, PMSF, SDS and TX-100 on ACP activity was investigated. k_m, V_max, k_cat and k_cat/k_m of ACP for p-NPP were found to be 0.09 mM, 2.75 U/mL, 9.60 s^-1 and 106.67 s^-1 mM^-1, respectively. The biochemical properties of ACP from Opuntia megacantha Salm-Dyck cladodes provide novel features with other plant ACPs and basic knowledge of ACP in Opuntia species.

Keywords: Acid phosphatase; Biochemical characterization; Cactus; Cladodes; Opuntia megacantha Salm-Dyck; Purification.