Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns.
An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
Vovk
Irena
I
National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. irena.vovk@ki.si
Simonovska
Breda
B
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000201:enzymology*; D008970:Molecular Weight; D011096:Polygalacturonase / Q000737:chemistry / Q000302:isolation & purification*
D007527:Isoenzymes; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D011096:Polygalacturonase