A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

Summary An enzyme from Trichoderma harzianum dissolved the cell walls of a wide range of filamentous fungi belonging to Basidiomycotina, Ascomycotina, Deuteromycotina, and Zygomycotina and so could be used to make protoplasts. A lyophilized preparation of the Trichoderma enzyme had about 0.3 units/mg -1,3-glucanase activity and 0.36 units/mg chitinase activity. About twice as many protoplasts were produced from different species of fungi by a single treatment with this enzyme than with combined commercial enzymes. The greatest number of protoplasts could be produced from most of the fungi by incubation for about 2 h t 30°C, but the number was decreased by incubation for more than 4 h or by use of a higher dose of the enzyme. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had less proteinase activity and protoplasts were fairly stable with this product during incubation for 8 h. Protoplasts produced by the proteinase-reduced preparation of the Trichoderma enzyme from three fungi regenerated at about 1.8 times the rate of those produced by the original enzyme.