PURIFICATION AND CHARACTERIZATION OF THREEISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

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Abstract
Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purified to homogeneity from tomato (var. S. marzano). The isozymes were separated by affinity chromatography on Heparin-Sepharose column. They exhibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sulfate gel electrophoresis and of 35 kDa in gel-filtration chromatography in native conditions. The isoelectric points of all three isozymes were found to be higher than 9.3. The Kms calculated for the three isozymes were different toward citrus pectin used as substrate; one had a Km of 9.7 mM (by expressing the pectin concentration as mmoles/L of methoxy groups) and the other two had similar Kms of 3.0 and 2.6 mM, respectively. The isozyme having the higher Km for substrate was inhibited by citrus pectin (which had a degree of methylation of 70%) at concentrations higher than 5 mM, but no inhibition was found using a pectin with a degree of methylation of 30% at concentrations up to 13 mM (i.e. 9 mg/ml) with a Km of 14.7 mM. Furthermore, this isozyme showed a more broad range of activity in a pH range 5–10 with respect to that exhibited by the other two isozymes. All three isozymes were found to be glycosylated, although to different extents.