Detection of beta-1,6-glucanase isozymes from Trichoderma strains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing gels.
The filamentous fungus Trichoderma produces, under specific growth conditions, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the antagonistic action of Trichoderma against a wide range of fungal plant pathogens. In this report we describe two different methods for the specific detection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase activity can be assayed in the gel by renaturation of the enzyme, incubation with an overlay agarose gel containing solubilized pustulan (a commercially available beta-1,6-glucan), followed by the staining of the agarose gel with Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be detected after incubation with solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium chloride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under different growth conditions.
Soler
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A
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Centro de Investigaciones Científicas Isla de la Cartuja, Sevilla, Spain.
de la Cruz
J
J
Llobell
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A
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development
D007527:Isoenzymes; D012967:Sodium Dodecyl Sulfate; D006026:Glycoside Hydrolases; C020105:endo-1,6-beta-glucanase