PURIFICATION TO HOMOGENEITY OF EXTRACELLULAR POLYGALACTURONASE AND ISOENZYMES OF PECTATE LYASE OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA BY COLUMN CHROMATOGRAPHY

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/l Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/l NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/l KCl gradient and PLI with a 3 mol/l KCl gradient followed by a 1.2 Mol/l PO4 buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pl values for PG, PLI and PII were 10.3, 10-3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.