Induction and regulation of cellulase synthesis in Trichoderma pseudokoningii mutants EA 3-867 and N 2-78

Abstract

Two mutants, EA 3-867 and N 2-78, with high cellulase yields were obtained from wild strains of Trichoderma pseudokoningii Rifai, 1096 and Mo 3, respectively, by mutagenic treatments with a linear accelerator, 60Co, u.v., nitrosoguanidine (NTG) and diethylsulphate (DTS). The mutants grew slowly to produce small colonies on agar plates with synthetic medium. On agar plates of peptone-yeast extract, the small colonies were as large as those of wild strains. The cellulase activities of these mutants in Koji extracts, shake flask culture filtrates, and enzyme preparations were markedly higher than those of their parents. The mutant N 2-78 reached quite high cellulase activity level when cultured for 60 h in shake flasks in a simple medium containing milled straw, wheat bran, mineral salts plus waste glucose molasses. The cellulase saccharifying activities on CMC, filter paper and cotton, were 255, 8.2 and 13.4 mg glucose/ml enzyme, respectively, or 11, 4.3 and 6 times more than those of its parent Mo 3 . The cellulase synthesis of EA 3-867 and N 2-78 was strongly induced by sophorose, isolated from pods of Sophora japonica L., and was inhibited by glucose, sugar phosphates, glycerol and organic acids. We conclude that cellulase synthesis of the mutants is regulated by catabolite repression as well as by induction. The increase in cellulase production by both mutants results from changes in the regulatory systems for cellulase synthesis, i.e. the mutants showed higher sensitivity to inducer and lower susceptibility to catabolite repression than did the wild types. A cellulase preparation of Trichoderma pseudokoningii Rifai N 2-78 induced by sophorose was fractionated by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography, selective inactivation and polyacrylamide gel electrophoresis. The components C 1(exo-β1,4-glucanase), C x(endo-β1,4-glucanase) and β-glucosidase were separated, and their molecular weights were estimated to be 67 000, 62 000 and 42 000 respectively. The homogeneity of C 1 was verified by polyacrylamide gel electrophoresis, immunoelectrophoresis and ultracentrifugal analysis. It is a glycoprotein and is rich in glycine, aspartic acid, threonine, serine and glutamic acid. The C 1 showed a strong synergistic action with C x in the degradation of cotton, Avicel and Walseth cellulose . A poly(A)-RNA, induced by sophorose in N 2-78 mycelium, was isolated by oligo(dT)-cellulose affinity chromatography.