This study developed a method for isolating and culturing protoplasts from the fungus Trichoderma reesei, a major producer of cellulase and β-glucosidase enzymes used in biofuel production. The researchers used Novozym 234, a commercially available enzyme preparation, to remove the cell wall from T. reesei mycelium under optimized conditions: 20-hour-old cultures, 0.9 M potassium chloride, and 18 hours at 28°C with gentle vibration. Over 95% of the resulting protoplasts remained viable and metabolically active. When protoplasts were induced with sophorose (a disaccharide inducer), they synthesized and secreted carboxymethylcellulase and β-glucosidase while remaining osmotically intact. Analysis using fast protein liquid chromatography revealed that both enzymes exist in multiple forms (at least seven peaks) even during early secretion, suggesting this multiplicity is not primarily generated by post-secretional cell wall modifications. The protoplasts represent a valuable system for studying cellulase secretion without interference from cell wall-associated enzymes.

Key findings

• Protoplasts from T. reesei strain QM 9414 were successfully isolated using Novozym 234 with >95% viability and retained metabolic activity including protein synthesis capability
• When induced with sophorose, protoplasts actively secreted both carboxymethylcellulase and β-glucosidase as confirmed by radiolabeled amino acid incorporation studies
• Multiple forms of both enzymes (at least seven distinct peaks) were detected in early secretion from protoplasts, indicating that enzyme multiplicity originates from biosynthesis rather than post-secretional modification
• The protoplast system eliminated cell wall interference in enzyme analysis, providing a clearer view of initially secreted enzyme forms compared to whole mycelium cultures