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 conditio ns, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the an tagonistic action of Trichoderma against a wide range of fungal plant patho gens. In this report we describe two different methods for the specific det ection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase ac tivity 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 wi th Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be d etected after incubation With solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium ch loride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under di fferent growth conditions. (C) 1999 Elsevier Science B.V. All rights reserv ed.