Alternative pathway for glucose production from cellulose using Trichoderma reesei QM9414 cellulase

Summary Evidence is presented which supports the view that two routes exist for the formation of glucose when cellulosic material is saccharified using T. reesei enzyme preparations. The first is via cellobiose and for the second, glucose appears to be formed by a route not involving cellobiose. The second route becomes more apparent when dealing with less crystalline cellulose. This should be considered when constructing strains to produce enzyme preparations for saccharification of less crystalline cellulose.     

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.     

Thermoinactivation of cellobiohydrolase I from Trichoderma reesei QM 9414

Irreversible thermoinactivation of cellobiohydrolase I from Trichoderma reesei has been analyzed at 70°C and pH 4.8. The time course of thermal inactivation and the dependence of the inactivation rates on protein concentration suggested that aggregation followed by precipitation was the main process leading to irreversible thermoinactivation. The enzyme activity was very resistant to 4 M urea which stabilized the enzyme against thermal inactivation. Deamidation of Asn/Gln residues and hydrolysis of peptide bonds were responsible for the loss of enzyme activity at long times of exposure at 70°C.     

Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414

beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.     

Mannanase activity of Endoglucanase III from Trichoderma reesei QM9414

Summary The cellulase and -mannanase activities of Endoglucanase III from Trichoderma reesei can be attributed to the same active site. Reversible and irreversible inhibitors affect similarly both activities. Galactomannan is a competitive substrate for a specific endoglucanase substrate. Km values are similar for mannan and carboxymethylcellulose but kcat values are ten times higher for the latter.     

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.     

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.     

Morphology of Trichoderma reesei QM 9414 in submerged cultures

The microscopic morphology of Trichoderma reesei QM 9414, growing in submerged culture, was studied by image analysis. The morphology was characterized by the total hyphal length, the total number of tips, the number of actively growing tips, and the length of the main hypha. To describe the growth of a single mycelium a simple model is set-up. The main features of the model are: (1) saturation type kinetics for the tip extension of the individual branches within the mycelium; and (2) random branching with a frequency function, which is proportional to the total hyphal length. The model is used to simulate a population of mycelia, where spore germination is described with a log-normal distribution. From the simulation of the population, the average properties of the mycelia, e.g., the average total hyphal length, are calculated, and by fitting the model to experimental data the model parameters are estimated. Finally, the distribution function with respect to the mycelia properties, that is, number of tips and total hyphal length, is calculated, and it corresponds well with the experimental determination of the distribution function. (c) 1995 John Wiley & Sons Inc.     

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.     

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.     

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.     

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.     

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).     

Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant

Summary The secretion of multiple forms of cellulolytic enzymes by a Trichoderma reesei QM 9414 selectant exhibiting high protease activity (T. reesei QM 9414/A 30) was investigated using monoclonal, domain-specific antibodies against cellobiohydrolase (CBH) I, CBH II and -glucosidase, and a polyclonal antibody against endoglucanase I. The pattern of appearance of these proteins was followed during growth of the fungus on Avicel cellulose, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting/immunostaining. Evidence was obtained that, at late cultivation stages, CBH I and II became partially modified to lower molecular weight components, whereas -glucosidase and endoglucanase I appeared to remain largely intact. Modification of CBH I appeared to commence from the carboxy-terminal AB region, whereas CBH II appeared to become modified both from the amino- (ABB') and the carboxy-terminal. Evidence for a protease activity that modifies the already truncated cellobiohydrolases in the culture filtrate was obtained. These results show that proteolysis at late culture stages may contribute to the multiplicity of cellulases found in T. reesei culture fluids. Initial proteolytic cleavage of CBH I and II may, however, involve an unusual protease not detectable by the azocasein method.Offprint requests to: C. P. Kubicek     

Chemical mechanism of beta-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters.

The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.