Summary: Protoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of by enzymic digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of tested. Treatment with digestive juice of the snail required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of β-glucuronidase and chitinase were determined. Electron microscopy of showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90 % of the protoplasts produced by this method were viable as assessed by vital staining with Janus Green B.


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