Bland S. Montenecourt

Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations

SummaryThe cellulases produced under pH controlled fermentation conditions with 5% Solka Floc and cornsteep liquor as substrates by Trichoderma reesei wild type QM6a and two mutants, Rut-C30 and RL-P37, have been separated by isoelectric focusing in polyacrylamide gels. The total complement of secreted proteins of the two mutants was distinct from the parent. However, the number and isoelectric points of the various enzymes in the cellulase complex were unchanged in the mutants. All secreted proteins stained with Schiffs reagent which indicated they were glycoproteins. One mutant, Rut-C30, exhibited a dramatic shift in the CBH I proteins during the course of the fermentation. RL-P37 showed a two-fold increase in the specific activity of both the total cellulase complex and endoglucanase. In addition a productivity on the order of 100 IU/l/h was achieved. Co-produced with the cellulases were at least two acid proteases with differential activity towards azocoll and azocasein.

Cellulase secretion from a hyper-cellulolytic mutant of Trichoderma reesei Rut-C30

Two strains of Trichoderma reesei, wild type QM6a and mutant Rut-C30, were grown in meida containing an inducer, insoluble crystalline cellulose (Avicel PH101), as carbon source for 11 days. The cell growth, expressed as myceliar protein content, of Rut-C30 was 4–5 times higher than QM6a. The lack of ultrastructural disorganization, and absence of intracellular enzyme release into the growth medium, indicated that none of these two strains had undergone any significant autolysis during the entire growth phase. Cellulase activities, mainly endoglucanase, cellobiase and filter paper degrading activity (disc) were enhanced in Rut-C30 cells. A major change was observed in the endoglucanase activity which was 30 times higher in Rut-C30 than QM6a, whereas, both β-glucosidase and disc activities were 3 times enhanced in Rut-C30 compared to QM6a. In addition to synthesis, cellulase secretion was also enhanced in Rut-C30. Both the organisms contained same amounts of intracellular marker enzyme activities (e.g., inosine diphosphatase, thiamine pyrophosphatase, alkaline phosphatase). Finally, the enahncement of secretory activity of Rut-C30 was correlated with the proliferation of rough endoplasmic reticulum (RER) and increased phospholipid content. It appears that Rut-C30 is not only a hypercellulolytic but also a hypersecretor mutant.

Effects of tunicamycin on secretion and enzymatic activities of cellulase fromTrichoderma reesei

SummaryThe effects of tunicamycin, an inhibi-tor of N-asparagine linked glycosylation, on the synthesis, secretion, and activities of the cellulases produced byTrichoderma reesei wild type QM6a and hypersecrefing mutant RL-P37 were studied. Neither the level of secreted cellutase nor the total amount of secreted protein was affected by the drug at a concentration (5 μg/ml) that slightly in-hibited growth. SDS-polyacrylamide gel electro-phoretic mobilities of proteins secreted during growth in tunicamycin were similar to those of proteins from control cultures that had their N-linked oligosaccharides removed by endoglycosi-dase H. Isoelectric focusing patterns of secreted proteins were also altered by growth in the pres-ence of tunicamycin. All of the bands stained with Schiff’s reagent, indicating that the secreted cellu-lases contained O-linked oligosaccharides in ad-dition to N-linked sugars. Endoglucanase activity in culture broths from tunicamycin grown mycelia was more thermolabile and protease-sensitive than the same activity from control cultures. Thus, N-asparagine linked oligosaccharides do not appear to be necessary forT. reesei cellulase secretion or activity, but do seem to contribute to the stability of the enzymes. The role of O-finked oligosaccharides is being investigated.

Detection of acrylic acid formation in Megasphaera elsdenii in the presence of 3-butynoic acid

SummaryThe formation of acrylic acid from lactic acid in the anaerobic rumen bacterium Megasphaera elsdenii was detected in the presence of 3-butynoic acid. While the major end products of lactic acid fermentation in the absence of the inhibitor were propionate, acetate, valerate, and butyrate, the presence of 3-butynoic acid led to the production of propionate, acetate, acrylate, and butyrate. An improvement in the chemical synthesis and purification of 3-butynoic acid was developed.

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

SummaryTemperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.

Protoplast formation and cell wall regeneration inClostridiumthermocellum

A protocol was developed for the production ofClostridiumthermocellum protoplasts and the regeneration of protoplasts into vegetative cells. Protoplasts were prepared by exposure of whole cells to lysozyme (125 ug/ml for 10 min. at 45 C). Protoplasts plated on regeneration agar reverted to walled cells with a frequency of up to approximately 0.3 % of input cells.