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Appl Environ Microbiol. 1985 September; 50(3): 615-622
Copyright © 1985, American Society for Microbiology. All Rights Reserved.

Activity Stain for Rapid Characterization of Pectic Enzymes in Isoelectric Focusing and Sodium Dodecyl Sulfate-Polyacrylamide Gels {dagger}

J. L. Ried and A. Collmer*

Department of Botany, University of Maryland, College Park, Maryland 20742

ABSTRACT

A system was developed for the rapid characterization of microbial pectic enzyme complexes and then tested on Erwinia chrysanthemi and Sclerotium rolfsii. Pectic enzymes in minute samples of crude culture filtrates were resolved by ultrathin-layer polyacrylamide gel isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then assayed with an ultrathin pectate-agarose overlay stained with ruthenium red. The simple procedure can be completed within 30 min after isoelectric focusing, can detect extremely low levels of pectate lyase (6.4 x 10–6 µmol of product per min), and is sufficiently sensitive to determine the pectate lyase isozyme profile of a single bacterial colony with a diameter of 4 mm. Pectate lyases and polygalacturonases can be distinguished by altering buffer conditions in the overlays. The assay system revealed additional isozymes not resolved by classical techniques and generally corroborated the previously published isoelectric points and molecular weights of the pectate lyase isozymes and exo-poly-{alpha}-D-galacturonosidase produced by E. chrysanthemi and the endopolygalacturonase and exopolygalacturonase produced by S. rolfsii.

FOOTNOTES

* Corresponding author.

{dagger} Scientific article no. A-4160, contribution no. 7145, of the Maryland Agricultural Experiment Station.

Appl Environ Microbiol. 1985 September; 50(3): 615-622
Copyright © 1985, American Society for Microbiology. All Rights Reserved.



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