Novozym 234, a commercially available enzyme from Trichoderma harzianum, has been used to prepare protoplasts from Trichoderma reesei QM 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 [14C]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 107 protoplasts ml–1. 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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Kolar et al. (1985) studied this question.