Novozym 234, a commercially available enzyme from , has been used to prepare protoplasts from 9414. Optimal conditions were: 20 h old mycelium, 0.9 M-KCl as the osmotic stabilizer. 0.1% (w/v) Novozym 234 and 18 h incubation at 28°C. To prevent damage to the protoplasts during isolation the incubation mixtures were agitated by vibration, so avoiding shaking or stirring. More than 95% of the protoplasts were viable and were also metabolically intact as shown by their ability to take up [C]leucine and incorporate it into cellular protein. Freshly prepared protoplasts could be induced by sophorose to produce and secrete carboxymethylcellulase and β-glucosidase. Optimal conditions were: 0.9 M-KCl as osmotic stabilizer, phosphate buffer pH 6.0, 7 mM-sophorose and 10 protoplasts ml. Separation of the secreted proteins by fast protein liquid chromatography revealed at least seven peaks of carboxymethylcellulase activity and β-glucosidase activity, indicating that some multiplicity of the forms of these enzymes is already present at the secretion stage.


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