Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation

A rapid and simple method was developed, using perfusion chromatography med ia, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating p ectinases present in a commercial tomato enzyme, preparation. Pectinase act ivities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the co lumn with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activit y. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. Th e PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PM E (tentatively identified as a "ubiquitous-type" isoform), and a pectin ace tylesterase. The later enzyme has not been reported previously in tomato. T his method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional propert ies.