Polygalacturonase (PG) secretion was analysed from five independent isolates of Botrytis cinerea grown in minimal liquid media enriched with pectin as the only carbon source. To acquire information about the biochemical characteristics of the PGs secreted in vitro, we combined enzyme-activity assays with Western blot analysis and isoelectro-focusing (IEF). All isolates secreted at least four PGs isozymes with a similar electrophoretic patterns, easily distinguishable by western blots. IEF enabled the isoelectric point of the PGs for each isolate to be determined. Wide variability in isoelectric point was observed between the PGs of each isolate, ranging from above pH 9.0 to below pH 5.0. For one of the isolates, each PG secreted in vitro was further purified by chromatofocusing and analysed for endo/eso-polygalacturonase activity by thin layer chromatography. This combination of analytical techniques and enzyme assays provided a view of the pool of PGs produced by B. cinerea and defined the biochemical features of each isozyme.

Biochemical characterisation of polygalacturonases from five different isolates of Botrytis cinerea

De Martinis, Domenico;Benvenuto, Eugenio
2002

Abstract

Polygalacturonase (PG) secretion was analysed from five independent isolates of Botrytis cinerea grown in minimal liquid media enriched with pectin as the only carbon source. To acquire information about the biochemical characteristics of the PGs secreted in vitro, we combined enzyme-activity assays with Western blot analysis and isoelectro-focusing (IEF). All isolates secreted at least four PGs isozymes with a similar electrophoretic patterns, easily distinguishable by western blots. IEF enabled the isoelectric point of the PGs for each isolate to be determined. Wide variability in isoelectric point was observed between the PGs of each isolate, ranging from above pH 9.0 to below pH 5.0. For one of the isolates, each PG secreted in vitro was further purified by chromatofocusing and analysed for endo/eso-polygalacturonase activity by thin layer chromatography. This combination of analytical techniques and enzyme assays provided a view of the pool of PGs produced by B. cinerea and defined the biochemical features of each isozyme.
Polygalacturonases;Botrytis cinerea
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.12079/360
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