Showing papers on "Trichoderma reesei published in 1999"

Comparison of Different Methods for the Detoxification of Lignocellulose Hydrolyzates of Spruce

Abstract: This study describes different detoxification methods to improve both cell growth and ethanol production by Baker's yeast, Saccharomyces cerevisiae. A dilute-acid hydrolyzate of spruce was used for the all detoxification methods tested. The changes in the concentrations of fermentable sugars and three groups of inhibitory compounds—aliphatic acids, furan derivatives, and phenolic compounds—were determined and the fermentability of the detoxified hydrolyzate was assayed. The applied detoxification methods included: treatment with alkali (sodium hydroxide or calcium hydroxide); treatment with sulfite (0.1% [w/v] or 1% [w/v] at pH 5.5 or 10); evaporation of 10% or 90% of the initial volume; anion exchange (at pH 5.5 or 10); enzymatic detoxification with the phenoloxidase laccase; and detoxification with the filamentous fungus Trichoderma reesei. An ion exchange at pH 5.5 or 10, treatment with laccase, treatment with calcium hydroxide, and treatment with T. reesei were the most efficient detoxification methods. Evaporation of 10% of the initial volume and treatment with 0.1% sulfite were the least efficient detoxification methods. Treatment with laccase was the only detoxification method that specifically removed only one group of the inhibitors, namely phenolic compounds. Anion exchange at pH 10 was the most efficient method for removing all three major groups of inhibitory compounds; however, it also resulted in loss of fermentable sugars.

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei.

Abstract: A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.

The Enzymatic Activity of Fungal Xylanase Is Not Necessary for Its Elicitor Activity

Abstract: Fungal xylanases from Trichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.

Dynamic Interaction of Trichoderma reesei Cellobiohydrolases Cel6A and Cel7A and Cellulose at Equilibrium and during Hydrolysis

Abstract: The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.

Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase

Abstract: A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei

Abstract: The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.

Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

Abstract: The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

Abstract: β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain.

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

Abstract: The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly.

Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Abstract: Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism The P pastoris rCBH I was recognized by MoAb raised to T reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T reesei enzyme could be isolated chromatographically The P pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (ie, pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism We conclude that P pastoris may not serve as an adequate host for the site-directed mutagenesis of T reesei CBH I

Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37.

Abstract: The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performances of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model as opposed to kinetic models. Using the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a "macromodeling" technique to rapidly develop dynamic models of a process.

Effects of temperature on the cellulose binding ability of cellulase enzymes

Abstract: The effects of high temperatures on catalytic activity and binding abilities of crude Trichoderma reesei cellulases in solution and adsorbed to a cotton fabric were studied. Above optimum temperature of 508C, catalytic activities were severely diminished but the binding behaviour was not found to be adversely affected. In order to verify possible applications of cellulases adsorbed to cotton fabrics as anchors for textile finishing purposes, we also checked the binding abilities after ironing. Previous ironing of cellulase adsorbed fabrics increased dyeability with an acid dye, but dye fastness was poor. Desorption of cellulases from cotton fabrics increased from pH 5 to pH 10. Dry ironing of fabrics resulted in less desorption, whereas wet ironing inhibited desorption at pH 5 and only 11% of protein were desorbed at pH 10. Ironing of the fabrics diminished enzyme activity of desorbed cellulases. Wet ironing resulted in complete denaturation of the proteins and no cellulolytic activity was found. The presence of water during thermal treatment of cellulases was found to be essential for complete denaturation and unfolding of the proteins. Dry heat only resulted in partial denaturation. Fluorescence measurements of cellulases adsorbed to cotton fabrics showed after ironing a significant shift in tryptophan fluorescence to higher wavelengths. This indicates unfolding and denaturation of the enzymes and revelation of more hydrophobic amino acids to the surface, which enables increased hydrophobic interactions with the fabric. q 1999 Elsevier Science B.V. All rights reserved. .

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure

Abstract: Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

Wastepaper Hydrolysate as Soluble Inducing Substrate for Cellulase Production in Continuous Culture of Trichoderma reesei

Abstract: The enzymatic hydrolysate of wastepaper was evaluated for its cellulase-inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 °C, and typical medium C:N ratio, the apparent cell yield constant was found to be 0.76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h -1 . The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4-fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase-inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h -1 ), the specific cellulase productivities of the hydrolysate-supported and the sophorose-induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose-induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate-supported systems increased from 2.2 FPU/g‚ ha tD ) 0.012 h -1 to 12.2 FPU/g‚ ha tD ) 0.122 h -1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.

Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

Abstract: Cellulose and chitin are composed of 1,4-β-linked pyranose units (in chitin, the C-2 hydroxyl groups have been replaced by N-acetyl residues) whose glycosidic bonds are hydrolyzed by cellulases and chitinases, respectively. Generally, these enzymes exhibit multidomain structures. The gram-negative bacterium Erwinia chrysanthemi is a plant pathogen that produces an endoglucanase, referred to as Cel5 (formerly known as endoglucanase Z). Cel5 consists of an N-proximal 287-amino-acid catalytic domain, a 35-amino-acid linker region, and a 62-amino-acid cellulose-binding domain (CBD). Catalytic domains and CBDs have been grouped into families on the basis of sequence homology (13, 16, 33). The catalytic domain of E. chrysanthemi Cel5 is in glycoside hydrolase family 5, and CBDCel5 is in CBD family V. The three-dimensional structure of CBDCel5 has been solved by nuclear magnetic resonance (NMR) spectroscopy (8). Other CBD structures determined by NMR spectroscopy are those of (i) family I CBDCBH1 from Trichoderma reesei cellobiohydrolase I (20, 24), (ii) family I CBDEG1 from T. reesei endoglucanase I (25), (iii) family II CBDCex from Cellulomonas fimi xylanase-glucanase (38), and (iv) family IV CBDN1 from C. fimi β-1,4-glucanase (18). The structures determined by X-ray crystallography are (i) family IIIa CBDCip from the scaffoldin subunit of Clostridium thermocellum (35) and (ii) family IIIc CBDE4 from Thermomonospora fusca endo/exocellulase E4 (31). CBDCel5 exhibits a boot-shaped structure based mainly on a triple antiparallel β-sheet perpendicular to a less-ordered summital loop (see Fig. ​Fig.1).1). There are five amino acids (Asp17, Trp18, Glu27, Trp43, and Tyr44) that potentially interact with the glucose chain of cellulose and contribute directly to the binding. In the present work, we have constructed a series of CBDCel5 mutants in which these amino acids were independently replaced by a functionally neutral alanine residue. FIG. 1 (a) Three-dimensional structure of CBDCel5. The residues that were mutated have been highlighted. (b) Backbone structure of CBDCel5. The highlighted residues represent the region that was deleted in the mutant Cel5ΔD17-P23. Additionally, we investigated the structural role of residues present in the turn of the boot-shaped model (cisPro11, Trp13). Also, the less-ordered loop region was investigated (Gln22Ala, Cel5ΔAsp17-Pro23). The region covering residues 29 to 61 corresponds to the β-sheet core of CBDCel5 and exhibits homology with sequences Yheb1 to -5 of an Escherichia coli hypothetical protein (8). Yheb1 to -5 are repeating domains of a 94-kDa protein in the Bfr-TufA intergenic region (1). In this paper, we describe the cellulose- and chitin-binding properties of the E. coli Yheb1 protein.

Enzyme-catalyzed saccharification of model celluloses in the presence of lignacious residues.

Abstract: Experiments were designed to determine the relevance of enzyme partitioning, between the cellulose and non-cellulose components of pretreated biomass, with respect to rates of cellulose saccharification in a typical biomass-to-ethanol process. The experimental system included three cellulose preparations (differing in physicochemical properties): a representative lignin-rich noncellulosic residue (prepared from dilute acid-pretreated switchgrass), an acid-extracted lignin preparation, and a complete Trichoderma reesei cellulase preparation. Enzyme-reactor conditions were typical of those commonly used in biomass-to-ethanol studies. The results were found to be dependent on both the lignin and cellulose preparations used. The noncellulosic lignacious residue, when supplemented at up to 40% (w/w) in cellulose-cellulase reaction mixtures, had little effect on rates and extents of cellulose saccharification. Overall, the results suggest that enzyme partitioning between cellulose and the noncellulosic component of a pretreated feedstock is not likely to have a major impact on cellulose saccharification in typical biomass-to-ethanol processes.

Saccharification of paper products by cellulase from Penicillium funiculosum and Trichoderma reesei

Abstract: Used paper products contribute largely towards organic-based waste produced and dumped by the world population. Cellulose, a structural component of paper materials, can be hydrolysed into glucose by a multi-component enzyme system called cellulase. Cellulase from Penicillium funiculosum and Trichoderma reesei were applied in the saccharification of paper products such as foolscap paper, filter paper, newspaper and office paper as well as microcrystalline cellulose. Foolscap paper showed the strongest susceptibility towards enzymatic hydrolysis with both enzymes. With an enzyme concentration of 10.0 mg/mL for each cellulase system the strongest synergistic action was observed at a combination of 1:1 (m/m) during saccharification of all cellulose materials. The individual enzyme performance as well as their synergistic actions showed different rates of hydrolysis during degradation of the investigated cellulose substrates.

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli.

Abstract: Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 (μ.mol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 μ.mol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively. Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.

Acid protease from Trichoderma reesei : limited proteolysis of fungal carbohydrases

Abstract: Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium.

Enzymatic Degradation of Cellulose-Based Materials

Abstract: The biodegradability of cellulose-based materials was compared in the standard Sturm test and by enzymatic hydrolysis. Trichoderma reesei culture filtrate, the purified enzymes endoglucanase I and II from T. reesei, and β-glucosidase from Aspergillus niger were used in the experiments. The unpurified Trichoderma reesei culture filtrate was found to contain a mixture of enzymes suitable for cellulose degradation. However, when purified enzymes were used the right balance of the individual enzymes was necessary. The addition of β-glucosidase enhanced the enzymatic hydrolysis of cellulose materials when both culture filtrate and purified enzymes were used. In the Sturm test the biodegradability of most of the cellulose materials exceeded 70% carbon dioxide generation, but, in contrast, the biodegradability of the highly substituted aminated cellulose and cellulose acetate was below 10%. The results concerning enzymatic hydrolysis and biodegradability were in good agreement for kraft paper, sausage casing, aminated cellulose, and cellulose acetate. However, diverging results were obtained with cotton fabric, probably as a result of its high crystallinity.

Hydrolysis of steam-pretreated lignocellulose: synergism and adsorption for cellobiohydrolase I and endoglucanase II of Trichoderma reesei.

Abstract: The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40°C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4°C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40°C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40°C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.

Adsorption and Activity of Trichoderma reesei Cellobiohydrolase I, Endoglucanase II, and the Corresponding Core Proteins on Steam Pretreated Willow

Abstract: The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBH I. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning.

Abstract: An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.

An α-l-Arabinofuranosidase from Trichoderma reesei Containing a Noncatalytic Xylan-Binding Domain

Abstract: l-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced α-l-arabinofuranosidase (α-AF) activity. An α-AF induced by l-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for p-nitrophenyl-α-l-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

Inhibition of cellobiohydrolases from Trichoderma reesei. Synthesis and evaluation of some glucose-, cellobiose-, and cellotriose-derived hydroximolactams and imidazoles.

Abstract: The lactam 16, the hydroximolactams 8, 20, 23, and 27, and the imidazole 32 were prepared following known methods. They were tested together with the known tetrazole 35 and the hydroximolactams 2 and 36 as inhibitors of the cellobiohydrolases Cel7A and Cel6A from Trichoderma reesei. Cel7A is only weakly inhibited by these compounds. Comparing their inhibitory activity evidences the importance of occupying subsites +1 and +2. The results strongly suggest that the shape of none of the variants of the lactone-type inhibitor motif embodied by these inhibitors is complementary to the subsite −1, i. e., analogous to the transition state. Cel6A is rather strongly inhibited by the cellobiose analogues 20, 23, and 32, and by the cellotriose analogue 27. Their relative inhibitory activities evidence that binding at subsite −2 depends upon the shape of the moiety occupying subsite −1. There is only a small difference between the inhibition by the hydroximolactams 20 and 23, which may be (partially) protonated by the catalytic acid of either anti- or syn-protonating glycosidases, and the imidazole 32, which can only be protonated by anti-protonating glycosidases. The results strongly suggest that shape requirements must be met by glycosidase inhibitors before they can be used to characterize the proton trajectory of glycosidases.