Purification and Some Properties of Two Polyphenol Oxidases from Bartlett Pears

Abstract

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K(m) values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V(max) and K(m) values for oxygen and chlorogenic acid were 103 mumoles O(2) uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K(m) values for chlorogenic acid were independent of pH from 3 to 7; the V(max) values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K(i) values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K(i) values of 0.04 and 0.11 mm for enzymes A and B, respectively.