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Biochemical characterization of polygalacturonases from five different isolates of Botrytis cinerea

Published online by Cambridge University Press:  08 October 2002

Dimitre PASHKOULOV ,
Irene GIANNETTI ,
Eugenio BENVENUTO  and
Domenico DE MARTINIS
Dimitre PASHKOULOV
Affiliation:
ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, C.P. 2400, 00060 Roma, Italy.
Irene GIANNETTI
Affiliation:
ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, C.P. 2400, 00060 Roma, Italy. Present address: European Institute of Oncology, Milan, Italy.
Eugenio BENVENUTO
Affiliation:
ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, C.P. 2400, 00060 Roma, Italy.
Domenico DE MARTINIS
Affiliation:
ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, C.P. 2400, 00060 Roma, Italy.
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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.

Type
Research Article
Information
Mycological Research , Volume 106 , Issue 7 , July 2002 , pp. 827 - 831
Copyright
© The British Mycological Society 2002

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