Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of -glucosidase upon induction with methyl--d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.     

Autopolyploid formation of Trichoderma reesei QM9414 by colchicine treatment

Conidia of Trichoderma reesei QM9414 were treated with colchicine. Nuclei in colchicine-treated conidia enlarged. When the concentration of colchicine or the treatment time with colchicine increased, the diameter of nuclei became larger. Colchicine-treated conidia generated sectors on a medium containing benomyl. Some sectors formed many conidia or could not produce clear zones on the plate assay medium for cellulase production. According to the DNA assay of conidia, colchicine-treated strains were autopolyploid.     

Action of Trichoderma reesei QM9414 and C30 cellulase on substrates with varying crystallinity

The action of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations from Trichoderma reesei QM9414 and C30 has been compared on Sigmacell, Solka Floc and alkali-treated bagasse in the presence and absence of added d-glucose and cellobiose. On the basis of equal filter paper activity the two preparations acted similarly on the two cellulosic substrates, while in the case of alkali-treated bagasse the C30 preparation gave greater d-glucose release. The relative levels of cellobiose produced from alkali-treated bagasse suggests that the non-cellobiose route was more important in d-glucose release by the C30 preparation compared to the QM9414 preparation.     

Multiple pH and temperature optimum of extracellular xylanase from Trichoderma reesei QM 9414

The rate of total extracellular xylanase production in Trichoderma reesei, QM 9414, system was affected by temperature and pH. In vitro studies with xylanase showed different temperature optima for activity in presence and in absence of xylan as substrate. Similar behaviour was observed in the pH studies. A number of temperature and pH optima also suggested the multiple nature of xyalanase.     

Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

Abstract Using ferritin-conjugated antibodies as an electron microscopic marker, β-glucosidase was localized within the cell walls of the imperfect fungus Trichoderma reesei QM9414. With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chalcea , β-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane.     

Localization and release mechanism of cellulases in Trichoderma reesei QM 9414

Summary A significant increase in the extracellular yield of -glucosidase was observed when Trichoderma reesei QM 9414 was cultivated on a cellulose medium containing chitin. Measurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while -glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degrading enzymes (produced from Trichoderma) led to a release of -glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chitinase and other cell wall lytic enzymes which promote release of the intramural -glucosidase into the medium.     

Localization of carboxymethyl cellulase in the intergeneric fusants of Trichoderma reesei QM 9414 and Saccharomyces cerevisiae NCIM 3288

In order to convert cellulosic material to ethanol by single step process a chemofusion method has been followed between protoplasts of Trichoderma reesei, QM 9414, and the spheroplasts of Saccharomyces cerevisiae, NCIM 3288, in the author's laboratory. The fusion was a success and it was observed that endoglucanase was the key enzyme in the success of the fusion. In the present study, characterization of the fusants based on the endoglucanase synthesis, its localization and the distribution in the cells are described and compared with that of Trichoderma reesei, QM 9414, (wild type).     

Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of beta-glucosidase.

Sophorose has two regulatory roles in the production of cellulase enzymes in Trichoderma reesei: beta-glucosidase repression and cellulase induction. Sophorose also is hydrolyzed by the mycelial-associated beta-glucosidase. Repression of beta-glucosidase reduces sophorose hydrolysis and thus may increase cellulase induction.     

Production of cellulases by Trichoderma reesei QM 9414 in batch and fed-batch cultures on wheat straw

Trichoderma reesei QM 9414 was grown in batch fermentation on wheat straw pretreated by different methods as the sole carbon source. Cellulase production was maximal with NaOH treated wheat straw at a concentration of 10 g/l and an initial pH of 5.5. The addition of fresh straw produced an elongation of the exponential phase or the beginning of a new exponential phase when the additions were carried out at 50 and 120 h, respectively. Filter paper and carboxymethylcellulase activities decreased as an answer to the addition of wheat straw and the levels were regained at the end of fermentation. The decreases of activities were accompanied by the increases of soluble sugar levels, which decreased at the end of fermentation. β-glucosidase activity was stimulated by wheat straw addition at 50 h while not by addition at 120 h; however, at the end of the fermentation the levels of activities were both similar to control. The studies of pH stabilities of these enzymes allow assurance that the effect of the addition of wheat straw on the enzyme activities is not produced by the changes of the pH during the fermentation.     

Production of cellulases by Trichoderma reesei QM 9414 in fed-batch and continuous-flow culture with cell recycle

The scope in improving enzyme productivities from the cellulose fermentation process is examined in laboratory-scale fermentors. The maximum productivity (30 IU/liter hr) is attained in a continuous-culture process with cell recycle using modified medium containing 0.5% cellulose. Optimum dilution rate and recycle ratio are determined as 0.025 hr-1 and 1.2, respectively, for the process. The system is analyzed and steady-state equations for predicting enzyme protein concentrations in the fermentor are developed. In fed-batch cultures, slow addition of cellulose at high concentrations can improve enzyme productivity by as much as 33% over a batch process. The scope and results of using modified medium for cellulase production are also presented.     

Homologous constitutive expression of Xyn III in Trichoderma reesei QM9414 and its characterization

Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2?±?65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53 % of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5–8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.     

Bioconversion of hemicellulose: aspects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose

The growth of Trichoderma reesei QM9414 in shake flasks at 28 degrees C on hemicellulose substrates and bagasse resulted in rather low yields of hemicellulolytic enzymes (1.0-1.5 units/mL xylanase and 0.05-0.08 units/mL beta-xylosidase). The influence of pH on the synthesis of beta-xylosidase was greater than on the synthesis of xylanase. Both xylanase and beta-xylosidase showed optimal activity at pH 4-5 and 55-60 degrees C. Xylanase was stable at pH 2-10 but was heat labile and totally inactivated after 1 h at 65 degrees C. Enzyme stability towards heat could be increased in the presence of bovine serum albumin. The beta-xylosidase was more tolerant to heat, but stable over a pH range 2.5-6.0. The D-xylose inhibited both enzymes in a competitive manner. Hemicellulose (heteroxylan) was degraded to the extent of 30-40%within 24 h. The degree of hydrolysis decreased as the substrate concentration increased and increased with increased amounts of enzyme. Multiple enzyme doses resulted in increased saccharification in reduced times. The degree of hydrolysis was influenced by the amount of beta-xylosidase present in the hemicellulolytic enzyme preparation. The -;xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of beta-glucosidase in the saccharification of cellulose by cellulases.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

β-Glucosidase production by Trichoderma reesei QM9414 on wheat straw. Location and some kinetic features

In this paper we studied the conditions for the production of β-glucosidase from T. reesei QM9414 in batch cultures using milled and sieved wheat straw as sole carbon source. High β-glucosidase production in the presence of wheat straw, a more realistic substrate than commercial cellulose, was obtained. The influence of particle size of wheat straw on β-glucosidase production in cell-free, cell and cell-wall extracts was studied. The particle size of wheat straw notably influenced enzyme production in cell and extramycelial extracts but it was less important with respect to the cell wall bound enzyme. β-glucosidase production was studied along of the fermentation. The results suggest a close relation between β-glucosidase from cell extract and extramycelial broth; geneticin levels of inhibition of β-glucosidase biosynthesis in both fractions were similar, a fact that suggests a common origin for the enzyme. Kinetic parameters for β-glucosidase from cell free and cell extracts were V_max = 0.28 μmol/min/mg, K_M = 0.91 mM and V_max = 0.095 μmol/min/mg, K_M = 0.39 mM respectively. Kinetic parameters for β-glucosidase from cell-wall could not be calculated because experimental data did not fit the different monosubstrate equations.     

Enhanced production of cellulase,hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30)

The Production of cellulases and Hemicellulases was studied with Trichoderma reesei Rut C-30, This organism produced, together with high cellulase activities, considerable amounts of xylanases and β-glucosidase. Three cellulose concentration (1, 2.5, and 5.0%) were examined to determined the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. The effect of temperature, pH, Tween-80 concentration, carbon sources, and substrate concentration on the ration of mycelial growth and extracellulose enzyme production are described.