Cellulase and related enzyme activity in protoplasts and mycelium of filamentous fungi.

Production of cellulase, pectinase and xylanase was investigated using culture filtrates from selected mesophilic (Aspergillus niger, Penicillium funiculosum, Penicillium janthinellum, Penicillium ochrochloron, Trichoderma viride), thermotolerant (Aspergillus fumigatus) and thermophilic (Geosmithia emersonii, formerly Talaromyces emersonii; the perfect stage of Penicillium emersonii) fungi. The fungi were grown on Acetobacter xylinum cellulose pellicles and soluble carboxymethyl cellulose and their enzyme complements were characterised under various growth conditions. The factors affecting the production and release of protoplasts from the closely related A. fumigatus and A. niger and P. ochrochloron and G. emersonii were investigated. Protoplast yields were found to be greatly dependent upon growth temperature, cultural conditions, mycelial age, mycelial concentration, incubation time, incubation temperature and lytic enzyme used. Greatest protoplast yields were obtained after 3 h incubation using 1 mg ml-1 Novozym TM 234 in 0.1 M 2-N-[Morpholino] ethane sulphonic acid (MES) buffer, pH 5.0 containing 0.6 M sodium chloride. Supplementation of the incubation medium with 0.03 M calcium chloride enhanced protoplast release in P. ochrochloron but not in the other three test fungi.Protoplasts from all four fungi were capable of regenerating a cell wall in both liquid and solid regeneration media. Regenerating protoplasts were shown to retain endo-B-1,4-glucanase, exo-B-1,4-glucanase and B-glucosidase activity. Attempts were made to cross related mesophilic with thermotolerant and/or thermophilic fungi using the technique of protoplast fusion. P. ochrochloron was crossed with G. emersonii and A. niger was crossed with A. fumigatus in an attempt to improve cellulase complement and stability. The test fungi were screened for resistance to a variety of heavy metal salts and commercial fungicides, and mutants resistant to Benlate and Mystox TM fungicides were obtained. These selection markers, together with an auxotrophic mutant of P. ochrochloron, were used to select out protoplast fusion products against parental types in the trial crosses.