PURIFICATION AND CHARACTERIZATION OF 3 ISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purifi ed to homogeneity from tomato (var. S. marzano). The isozymes were sep arated by affinity chromatography on Heparin-Sepharose column. They ex hibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sul fate gel electrophoresis and of 35 kDa in gel-filtration chromatograph y in native conditions. The isoelectric points of all three isozymes w ere found to be higher than 9.3. The K(m)s calculated for the three is ozymes were different toward citrus pectin used as substrate; one had a K(m) of 9.7 mM (by expressing the pectin concentration as mmoles/L o f methoxy groups) and the other two had similar K(m)s of 3. 0 and 2.6 mM, respectively. The isozyme having the higher K(m) 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 usin g a pectin with a degree of methylation of 30 % at concentrations up t o 13 mM (i.e. 9 mg/ml) with a K(m) of 14.7 mM. Furthermore, this isozy me showed a more broad range of activity in a pH range 5-10 with respe ct to that exhibited by the other two isozymes. All three isozymes wer e found to be glycosylated, although to different extents.