Showing papers on "Trichoderma reesei published in 1994"

The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.

Abstract: Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose. The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside. The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the plant legume lectins.

Enzymatic conversion of biomass for fuels production.

Abstract: Bioconversion for Production of Renewable Transportation Fuels in the United States: A Strategic Perspective Cellulase and Xylanase Systems of Thermotoga neapolitana Structure-Function Studies of Endo-1,4-*b-D-glucanase E2 from Thermomonospora fusca Role of Cellulose-Binding Domain of Cellobiohydrolase I in Cellulose Hydrolysis CelS: A Major Exoglucanese Component of Clostridium thermocellum Cellulosome Plant Endo-1,4-*b-D-glucanases: Structure, Properties, and Physiological Function Approaches to Cellulase Purification Cellobiose Dehydrogenase: A Hemoflavoenzyme from Phanerochaete chrysoporium Cellulase Production Technology: Evaluation of Current Status Cellulase Assays: Methods from Empirical Mathematical Models Components of Trichoderma reesei Cellulase Complex on Crystalline Cellulose: Three-Dimensional Visualization with Colloidal Gold Deposition of Metallic Platinum in Blue-Green Algae Cells Genetic Engineering Approaches for Enhanced Production of Biodiesel Fuel from Microalgae Microbial and Enzymatic Biofuel Cells Pretreatment of Lignocellulosic Biomass Bioconversion of Wood Residues: Mechanisms Involved in Pretreating and Hydrolyzing Lignocellulosic Materials Development of Genetically Engineered Microorganisms for Ethanol Production Kinetic Consequences of High Ratios of Substrate to Enzyme Saccharification Systems Based on Trichoderma Cellulase Pectin-Rich Residues Generated by Processing of Citrus Fruits, Apples, and Sugar Beets: Enzymatic Hydrolysis and Biological Conversion to Value-Added Products Silage Processing of Forage Biomass to Alcohol Fuel Conversion of Hemicellulose Hydrolyzates to Ethanol Anaerobic Digestion of Municipal Solid Waste: Enhanced Cellulolytic Capacity Through High-Solids Operation Compared to Conventional Low-Solids Systems Role of Acetyl Esterase in Biomass Conversion Metabolism of Xylose and Xylitol by Pachysolen tannophilus

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

Abstract: The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 183% at 18 A resolution The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel The beta-sheet structure is twisted, forming a large cleft on one side of the molecule The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft One structure was determined at pH 50, corresponding to the pH optimum of XYNII The second structure was determined at pH 65, where enzyme activity is reduced considerably A clear structural change was observed, especially in the position of the side chain of Glu177 The observed conformational change is probably important for the mechanism of catalysis in XYNII

Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction.

Abstract: The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented.

A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast.

Abstract: Summary A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesel and isolated a new β-1,4-endoglucanase gene on plates containing β-glucan. This gene, eg15, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the iinker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major celluiase CBHI from T. reesei. The catalytic core of EGV is unusually small for a ceiiulase and represents a new family of ceilulases (Family K) and of glycosyl hydrolases (Famlly 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic ciuster anaiysis.

Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals.

Abstract: Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E. coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pki1 (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively. Transformation frequencies of over 1,800--2,500 transformants/micrograms DNA were obtained, which is a 15--20-fold increase over that with pAN7-1, which contains hph between A. nidulans expression signals. Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pki1 promoter and the cbh2 terminator integrated into the genome. Evidence for preferentially ectopic integration is given.

Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis.

Abstract: Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose.

Abstract: Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohydrolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30--90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased withmore » increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I.« less

Mixed culture solid substrate fermentation for cellulolytic enzyme production

Abstract: Cellulolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864. Optimal cellulose and xylanase levels of 4 IU/g dry weight (DW) and 180 IU/g DW, respectively, were achieved in 120 h-fermentation when T. reesei, inoculated at 0 h, was followed by the inoculation of A. niger at 48 h.

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

Abstract: A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia.

Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a beta-1,4-exocellobiohydrolase analogous to Trichoderma reesei CBH II.

Abstract: Summary The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.

A new thermostable endoglucanase, Acidothermus cellulolyticus E1

Abstract: A new thermostable endoglucanase,Acidothermus cellulolyticus E1, and another bacterial endoglucanase, E5 fromThermomonospora fusca, each exhibit striking synergism with a fungal cellobiohydrolase (Trichoderma reesei CBH I) in the saccharification of microcrystalline cellulose. In neither case did the ratio of endoglucanase to exoglucanase that demonstrated maximum synergism coincide exactly with the ratio that actually released the maximum quantity of soluble sugar for a given total cellulase loading. The difference between the two ratios, after significant hydrolysis of the substrate, was considerably larger in the case ofA. cellulolyticus E1. For both endoglucanase pairings with CBH I, the offset between the ratio for maximum synergism and the ratio for maximal soluble sugar production was found to be a function of digestion time.

Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies.

Abstract: Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a “full-value” cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. © 1994 John Wiley & Sons, Inc.

Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium.

Abstract: Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.

Saccharification, fermentation, and protein recovery from low‐temperature AFEX‐treated coastal bermudagrass

Abstract: Coastal bermudagrass was pretreated by a low-temperature ammonia fiber explosion (AFEX) process, which soaked the grass in liquid ammonia and then explosively released the pressure. Saccharifying enzymes were systematically applied to the AFEX-treated grass corresponding to low, medium, and high loadings of cellulase/hemicellulase (from Trichoderma reesei), cellobiase, glucoamylase, and pectinase. Three-day sugar yields linearly correlated with the logarithm of the cellulase loading. Supplemental enzymes (cellobiase, pectinase) caused upward shifts in the lines. The linearity and upward shifts are consistent with the HCH-1 model of cellulose hydrolysis. The hydrolysis sugars were converted to ethanol using yeast (Saccharomyces cerevisiae). The solid residues were treated with proteases to attempt recovery of valuable proteins. The low-temperature AFEX pretreatment was able o nearly double sugar yields. At the highest cellulase loadings (30 IU/g), the best reducing sugar and ethanol yields were 53% and 44% of the maximum potential, respectively. Protein recovery was, at most, 59% © 1994 John Wiley & Sons, Inc.

Morphological and macromolecular characterization of Hypocrea schweinitzii and its Trichoderma anamorph

Abstract: Morphological, cultural, and isozyme anal- yses were used to determine variation in the Tricho- derma anamorph of Hypocrea schweinitzii, a presumably unequivocal species of Trichoderma, and to assess whether the anamorph of H. schweinitzii can be as- signed to T. longibrachiatum, T reesei, or T. pseudoko- ningii. The results of these studies indicate that H. schweinitzii comprises at least three distinct and tax- onomically separable holomorph taxa that coincide with the geographical origin of the collections. The name H. schweinitzii can be applied to Northern Hemi- sphere and H. jecorina to tropical American collec? tions; while taxonomically distinct, no name was given to a Chinese collection. The holomorphs of H. schwei? nitzii, H. jecorina, and the Chinese collection showed little intraspecific variation in morphology of the teleo- morph or anamorph, or in cultural and isozyme char- acters. They exhibited no more variation than was noted in the ex type cultures of the named Trichoderma species. None of the named Trichoderma species co- incided with any of the Hypocrea species studied. It is concluded that Trichoderma species in general can be narrowly defined but that morphology alone might not suffiee to allow the identification of species. The syn-

Production of endo-1,4-β-glucanase and xylanase by Trichoderma reesei immobilized on polyurethane foam

Abstract: Endo-1,4-β-glucanase and xylanase were produced by Trichoderma reesei immobilized on polyurethane foam using lactose as the main carbon source. The most porous carrier was found to be the best of those tested. The nitrogen source and KH2PO4 concentration of the production medium had a marked effect on culture pH during the course of fermentation and, consequently, on xylanase activity. An increase in lactose concentration from 7 to 27 g/l resulted in an increase in endoglucanase activity (max. 730 U/ml), xylanase activity (max. 3350 U/ml) and filter paper activity (max. 3.0 FPU/ml).

Novel endoglucanase enzyme

Abstract: The present invention concerns a novel enzyme, EGV, having endoglucanase activity. The molecular weight of the enzyme is about 20 to 25 kDA and it is isolated from the fungus Trichoderma reesei. The invention also relates to a DNA sequence coding for the novel enzyme as well as vectors, yeast strains and fungal strains containing the DNA sequence. Furthermore, the invention concerns a method for isolating the DNA sequence coding for the novel enzyme and for constructing yeast and fungal strains which are capable of expressing endoglucanase. The invention also provides an enzyme product having endoglucanase activity and methods for enzymatically modifying lignocellulosic materials, in particular for modification or degradation of cellulose and/or β-glucan.

Enzymes as silage additives for grass–clover mixtures

Abstract: Seven laboratory-scale experiments were carried out to study the effects of cellulases/hemicellulases on silage fermentation of herbage from mixed swards of timothy (Phleum pratense), meadow fescue (Festuca pratensis) and red clover (Trifolium pretense) Enzyme-treated silage (approximately 3500 HEC units kg−1 grass) reached a low pH sooner, had lower end pH, contained less NH3-N and more lactic acid than did the untreated silage (P < 0·05) Applied with an inoculant, these effects were even stronger With easily ensiled crops (experiments 1, 2, 5 and 6) preservation was first of all improved by inoculation of lactic acid bacteria; however, for the low-sugar crops (experiments 3, 4 and 7) enzyme treatment was more significant The enzymes derived from Aspergillus spp gave more acetic acid than the enzymes from Trichoderma reesei The Trichoderma enzymes liberated 4·8 g WSC kg−1 FM gamma-irradiated grass during 60 d at pH 4·3 (P < 0·05) On average, for all silages enzyme treatment increased the sum of residual sugar and fermentation products by 3·7 g kg−1 FM (21 g kg−1 DM) compared with the silages not treated with enzymes (P < 0·001) Enzyme treatment increased the instantly degradable part of the feed, but total in sacco and in vitro digestibilities were not affected

Trichoderma reesei cellulases in the bleaching of kraft pulp

Abstract: In this work the effects of individual purified cellulases of Trichoderma reesei were studied in the enzyme-aided bleaching of kraft pulps. The cellobiohydrolases I and II, when used alone, had no positive effect on the bleachability of kraft pulps. The endoglucanase I (EG I), however, acted on pulp similarly to xylanases and with an enzyme dosage of 0.1 mg/g a clear increase in pulp brightness could be observed. Due to the unspecificity of this enzyme, the viscosity of the pulp was simultaneously decreased. Of the cellulases, EG II was clearly most detrimental in reducing the pulp viscosity. Hence, the action of purified cellulases of T. reesei on pulp as a substrate differs profoundly, and all cellulases are not detrimental to the pulp properties.