The purification and properties of peanut phytase and the identification of the myo-inositol phosphates from partial dephosphorylation of myo-inositol hexaphosphate by the enzyme

Abstract

Peanut phytase (meso-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8) was purified 17-fold over an extract of defatted peanut seeds by (NH₄)₂SO₄ fractionation. The pH optimum (5.0) and the apparent Michaelis constant (0.1M) were determined for the action of this phytase preparation on phytic acid. Substrate specificity studies, using this preparation, indicated a broad specificity for several phosphomonoester substrates. The pH optima for this phytase preparation for action on α- and β-glycerophosphate were found to be 5.6 and 5.5, respectively. Further fractionation of the (NH₄)₂SO₄ enzyme preparation by pH 5 precipitation, acetone and methanol fractionation yielded an enzyme preparation with a specific activity 150-times that of a peanut flour extract. During these fractionations a splitting of phytase activity into several fractions was noted. Substrate specificity studies on these fractions yielded no evidence for a phytic acid-specific enzyme preparation. Extracts of subcellular particles prepared from resting peanut seeds confirmed that phytase activity is concentrated in the aleurone grain fraction. Phytic acid was isolated from defatted peanut flour, hydrolyzed, and the inositol isolated. Comparison with an authentic standard of myo-inositol by melting point and infrared spectrometry of their acetate derivatives confirmed that peanut phytic acid is myo-inositol hexaphosphate. Large scale phytic acid hydrolyses were run using the (NH₄)₂SO₄ phytase preparation as the enzyme source. The myo-inositol phosphate intermediates from the reaction were isolated and their structure determined by periodate oxidation, acid-catalyzed phosphate migration across cis hydroxyls, and partial alkaline hydrolysis. Evidence is presented that the dephosphorylation of phytic acid by peanut phytase proceeds through the following intermediates: 1. postulated myo-inositol-1,2,3,5,6(1,2,3,4,5)-pentaphosphate; 2. myo-inositol-1,2,3,4(1,2,3,6)- and 1,2,5,6(2,3,4,5)-tetraphosphates; 3. myo-inositol-1,2,3- and 1,2,6(2,3,4)-triphosphates; 4. myo-inositol-1,2(2,3)-diphosphates; and 5. myo-inositol-2- and 1(3)-monophosphates.