Pectinesterases from mature, unripe peach fruit bind strongly to pectic polysaccharides

Contrary to previous findings, the level of the pectin de-esterifying enzyme, pectinesterase (PE; EC 3.1.1.11), is shown to be much higher in mature, green peach fruit than in ripe fruit. Aqueous buffers readily extracted three pectinesterase isoforms from ripe fruit but only a portion of the activity from mature, green fruit. In mature, green fruit extracts the enzyme precipitated when the ionic strength was lowered; consequently isoforms could not be recovered by ion exchange chromatography. In extraction residues from mature, green fruit, residual PE could be measured as active enzyme and, when denatured, could be detected by immunological techniques. Extraction of the enzyme was enhanced after digestion of the tissue with pectin lyase. The extracted enzyme fractionated as a large molecular weight complex rich in uronic acid, rhamnose, galactose and arabinose. After further digestion with endo-beta-1,4-galactanase, the enzyme was in two fractions of smaller size but with residual carbohydrate. When mature, green and ripe fruit tissue were co-extracted, the recovered activity was as predicted from independently extracted tissues demonstrating that enzyme activity was not influenced by inhibitors contributed by either tissue type. Isoforms known to be present in the ripe fruit were recovered from extracts of the mixed tissues. It is concluded that PE in extracts of mature, green fruit has a strong association with pectic polymers and this has lead to its underestimation in previous studies. It is suggested that such an association with pectin polymers in vivo may regulate enzyme activity and enzyme turnover.