Changes in the regulatory mechanism of cellulase s...
Two mutants，EA3-867 and N2-78，showed much higher cellulase yields than wild parents in Koji extracts，shake flask culture filtrates，and enzyme preparations.The mutant N2-78 showed a quite high activity level when cultured in shake flasks on a simple medium containing milled straw，wheat bran，mineral salts plus waste glucose molasses for 60 h，and the highest saccharifying activities of cellulase on CMC，filter paper and cotton reached 255，8.2 mg and 13.4 mg glucose/ml，respectively，or 11，4.3and 6 times more than those ofits parent Mo3（see Table 4）.Crude N2-78 cellulase preparation of a 1%（w/v）enzyme solution in 0.01 M citrate buffer，pH=4.8，in a Warburgshaker（80 strokes/min）at 50℃was able to degrade filter paper（Whatman no.1，1 cm2）into fine fibres after only 9 min.
Table 4 Comparison of activities of cellulase from mutants and wild strains of Trichoderma in flask cultures［1］
Seven ml mycelium seed suspensions were inoculated into 100 ml of a rich straw medium in 500 ml flasks，and cultured in a rotary shaker（104 strokes/min）at 30℃for 60 h.The enzyme filtrates were used to assay cellulase activities.The rice straw medium consists of（%）：milled rice straw，1；wheat bran，0.5；（NH4）2 SO4，0.2；KH2 PO4，0.1；CaCO3，0.1；glucose molasses，1.The mycelium seed suspensions were obtained by culturing the spare suspensions of different strains on a seed medium at the same condition for 32 h.The seed medium was the same as ricestraw medium except that rice bran was used in place of wheat bran.CMC saccharifying activity，FP and cotton cellulases were assayed by the procedure suggested by Mandels，［17］and expressed as mg glucose produced per ml in 30 min，60 min and 24 h incubation，respectively.CM cellulase（liquefying activity）was determined by the decrease in CMC viscosity and expressed as units per ml per min.［1，20］
Morphologically，both mutants produced smaller colonies on agar plates containing synthetic medium or potato glucose medium，and grew more slowly than their parents.But on agar plates containing peptone-yeast extract medium，there were no differences in colony size among the strains.［8］
Besides growth and morphology，the kinetic characteristics and components of cellulase in the mutants and wild strains were also compared.The optimal pH and temperature for cellulase activities（CM cellulase，FP cellulase）were the same.［1］No differences in cellulase components were observed，at least in the patterns of polyacrylamide gel electrophoresis，except that the protein bands of cellulase of the mutants had a darker colour than that of their parents.［8］It is suggested that the changes in the mutants probably do not occur in the structural genes，but only in the regulatory genes which may be involved in higher yield of cellulase.
Since the regulatory systems of cellulase in Trichoderma consist of the induction and catabolite repression，the sensitivity of the mutants and parents to inducer（sophorose）and repressor（glucose）were compared in a washed mycelium test.Cellulase of the mutants could be induced by lower sophorose（10—100 times less than in the parents）（see Table 5），and were repressed by higher glucose（almost 10 times higher than in the parents）（see Table 6）.It is evident that the increase in cellulase production of both mutants，which have been shown to be partially derepressed，results from the change in induction as well as in catabolite repression.
Table 5 Comparison of sensitivity of washed mycelia of different strains of Trichoderma to sophorose induaion of cellulase［8］
（continued）
Mycelia：1096，2.4 mg/ml；EA3-867，1.9 mg/ml；Mo3，2.5 mg/ml；N2-78，1.7 mg/ml.Sophorose：10-4 M.
Table 6 Comparison of sensitivity of washed mycelia from different strains of Trichoderma to glucose repression［8］
Mycelia：1096，2.9 mg/ml；EA3-867，2.1 mg/ml；Mo3，2.5 mg/ml；N2-78，1.1 mg/ml.Sophorose：10-4 M.
