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 media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities 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 column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.
Savary
B J
BJ
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. bsavary@arserrc.gov
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010477:Perfusion / Q000379:methods*; D037121:Plant Lectins; D011096:Polygalacturonase / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000032:analysis
D007527:Isoenzymes; D010446:Peptide Fragments; D037121:Plant Lectins; D003208:Concanavalin A; D012965:Sodium Chloride; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D011096:Polygalacturonase