Isolation and enzymatic properties of a nonspecific acid phosphatase from Vigna aconitifolia seeds

Abstract

Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulfate fractionation and cation-exchange chromatography [carboxymethyl (CM) cellulose]. The enzyme was 228-fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 column showed that Mr of native enzyme was 58 kDa and denaturing PAGE demonstrated that it was made up of two subunits of 24 and 27 kDa. The enzyme showed its optimum activity at pH 5.0 and 60°C. It exhibited broad substrate specificity and showed a higher specificity constant for para-nitrophenyl phosphate, Na β-naphthyl phosphate, and adenosine monophosphate (AMP). Cu²⁺, Mo⁶⁺, Fe³⁺, phosphate, and fluoride ions were reported as strong inhibitors for the enzyme. Active site study for the enzyme demonstrated that tryptophan and aspartic acid may be important for the catalysis.