Purification and characterization of homogeneous acid phosphatase from nongerminated buckwheat (Fagopyrum esculentum) seeds

A buckwheat acid phosphatase (orlhophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was purified about 250-fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p-nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p-nitrophenyl phosphate were determined to be K-M = 76 mumol L-1 and k(cat) = 924 s(-1) at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.