Protoplast production and localization of cellulase in species of Aspergillus.

Toenhance the production of protoplasts the factors affecting the release of protoplasts from A. niger and A. fumigatus were investigated. Factors which strongly affected protoplast release were pH, temperature, concentration of the osmotic stabilizer and the concentration of calcium ions. The hydrolysis of the inner, chitin wall - layer was an important factor in ensuring optimal protoplast release. For this reason chitinase was included in the digestion medium.Carboxymethylcellulase (CMCase) was produced in the mycelium of A. fumigatus growing on ball milled cellulose (BMC) and on carboxy-methylcellulose (CMC). Actively growing mycelia released the enzyme into medium containing EMC but not into medium containing CMC.Sharp peaks of enzyme synthesis and activity were evident after an initial lag period. Supplementation of the culture medium with glucose considerably reduced this lag period.Localization procedures indicated that the enzyme occurred within the cell wall, in the periplasmic space and upon, or enclosed by, membranes. CMCase was released by the action of chitinase enzymes and by cold osmotic shock. It was also sedimented by high-speed centrifugation of cell homogenates from young mycelia. Older mycelia contained more soluble CMCase. Histochemical localization showed the enzyme to be present in cytoplasmic vesicles and in the cell wall. Triton X-100 acted to increase the release of CMCase in crude homogenates and in centrifuged fractions.Protoplasts regenerating in CMC medium released CMCase into the medium but retained little in the cytoplasm. Cycloheximide was found -to substantially reduce the production of CMCase by protoplasts and protoplasts from mycelium already producing CMCase were not predisposed to synthesise the enzyme.