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PubMed1993

Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei.

The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CBH I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1996

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169.

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.

Binding Sites↗

PubMed1988

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis.

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.

Amino Acid Sequence↗

PubMed1997

Role of four major cellulases in triggering of cellulase gene expression by cellulose in Trichoderma reesei.

The relative contributions of four major cellulases of Trichoderma reesei (1,4-beta-D-glucan cellobiohydrolase I [CBH I], CBH II, endo-1,4-beta-D-glucanase I [EG I], and EG II) to the generation of the cellulase inducer from cellulose were studied with isogenic strains in which the corresponding genes (cbh1, cbh2, egl1, and egl2) had been deleted by insertion of the Aspergillus nidulans amdS marker gene. During growth on lactose (a soluble carbon source provoking cellulase gene expression), these strains showed no significant alterations in their ability to express the respective other cellulase genes, with the exception of the strain containing delta cbh1, which exhibited an increased steady-state level of cbh2 mRNA. On crystalline cellulose as the only carbon source, however, significant differences were apparent: strains in which cbh2 and egl2, respectively, had been deleted showed no expression of the other cellulase genes, whereas strains carrying the cbh1 or egl1 deletion showed these transcripts. The delta cbh1-containing strain also showed enhanced cbh2 mRNA levels under these conditions. A strain in which both cbh1 and cbh2 had been deleted, however, was unable to initiate growth on cellulose. Addition of 2 mM sophorose, a putative inducer of cellulase gene expression, to such cultures induced the transcription of egl1 and egl2 and restored the ability to grow on cellulose. We conclude that CBH II and EG II are of major importance for the efficient formation of the inducer from cellulose in T. reesei and that removal of both cellobiohydrolases renders T. reesei unable to attack crystalline cellulose.

Cellulase↗

PubMed1991

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

Antibodies, Monoclonal↗

PubMed1996

Cloning of a gene encoding a putative carbon catabolite repressor from Trichoderma reesei.

We report the cloning and sequencing of a Trichoderma reesei genomic DNA clone that encodes a protein similar to the Aspergillus CREA, a carbon catabolite repressor. The deduced amino acid sequence predicts a zinc finger protein of 402 amino acids in length. The overall amino acid sequence of the T. reesei CREA shows about 52% similarity with Aspergillus CREAs.

Amino Acid Sequence↗

PubMed2000

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism.

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.

Acetylation↗

PubMed2000

[Studies on simultaneous production of chitosanase and chitosan degradation in situ with Trichoderma reesei in convoluted fibrous bed bioreactor].

The cells of Trichoderma reesei were immobilized on a roll of porous polyurethane foam sheet and packed in a bubble column bioreactor for simultaneous production of chitosanase and degradation of chitosan in situ. The average degree of polymerization could be regulated by reaction time. Under the repeated-batch process with 2% soluble chitosan at pH4.8, 28 degrees C, the activity of chitosanase for each batch was above 0.15 IU/mL, the average yield of reducing sugar as D-glucosamine reached 73%. The novel immobilized bioreactor system run stably and effectively in the successive 10 batches lasting 30 days without notable change in the activity and productivity.

Bioreactors↗

PubMed1993

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei.

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.

Amino Acid Sequence↗

PubMed1998

Isolation and identification of xylitol dehydrogenase gene from Trichoderma reesei.

A cDNA sub-library from the fungus Trichoderma reesei grown on xylan was constructed in S.cerevisiae recombinant strain H475 harboring a xylose reductase (XR) gene from Pichia stipitis. The xylitol dehydrogenase (XDH) gene was screened from sub-library on a SC selective medium in which xylose was used as a sole carbon source. The length of XDH gene, xdh1, was about 1.3 kb. Southern, Northern and Western blots were carried out, indicating that xdh1 has high affinity with T.reesei and the molecular weight of the xylitol dehydrogenase from T.reesei was about 40 kDa. The strain HX1 harboring both the XR gene from P.stipitis and the XDH gene from T.reesei was able to grow on the xylose medium and converted more than 90% of the xylose into xylitol, ethanol, and other by-products.

Cloning, Molecular↗

PubMed2005

Production of cellulase by Trichoderma reesei from dairy manure.

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.

Animals↗

PubMed1995

Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei.

Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei. The enzyme contains a discrete cellulose-binding domain (CBD), which increases its binding and activity on crystalline cellulose. We studied cellulase-cellulose interactions using site-directed mutagenesis on the basis of the three-dimensional structure of the CBD of CBHI. Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism. The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality. The data also suggest that the more hydrophobic surface is not directly involved in the CBD function. The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption. The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect. High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD.

Adsorption↗

PubMed2002

The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175.

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

Aspartic Acid↗

PubMed1996

Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.

Amino Acid Sequence↗

PubMed2026

Trichoderma reesei Nsd3 transcription factor: pleiotropic roles in development, stress response, secondary metabolism, and cellulase production.

Trichoderma reesei is known for its ability to secrete high amounts of cellulases, enzymes of fundamental importance in generating products from lignocellulosic biomass. Diverse signaling pathways and transcription factors (TFs) control the cellulolytic repertoire in T. reesei to ensure correct adaptation to the environment. Here, we analyzed RNA-Seq data and identified a new potential regulator of cellulase production in T. reesei: a novel TF named Nsd3, a homolog of NsdC from Aspergilli. Deletion of nsd3 reduced vegetative growth and conidiation on solid medium. Phenotypic characterization of the Δnsd3 strain showed that it is more sensitive to osmotic stress, but more resistant to cell wall and oxidative stresses. Our results showed that Nsd3 is a repressor of cellulase expression by directly regulating key genes in the cellulolytic pathway, an unreported role for this TF in fungi. Loss of nsd3 leads to a faster and more robust induction of cellulolytic genes, and higher cellulase and hemicellulase activities. Transcriptional profiling by RNA-Seq, chromatin accessibility profiling by ATAC-Seq, and protein-DNA interaction assays showed that sugar transporters are important targets of Nsd3 during cellulase expression regulation. Combined with microscopy and gene expression analyses, the ATAC-Seq data also highlighted Nsd3 as a central regulator of cell wall remodeling and organization. Furthermore, the transcriptomics also showed that Nsd3 regulates genes involved in secondary metabolism. These results showed that Nsd3 regulates several physiological processes and provide novel insights into the regulatory system of cellulases in T. reesei that can be used in the design of high-performance strains for biorefinery.IMPORTANCETrichoderma reesei is a key player in the production of hydrolytic enzymes for the degradation of lignocellulose biomass, and transcription factors are important targets for genetic engineering to construct cellulase-hyperproducing strains. Here, we identified the transcription factor Nsd3 and characterized its role as a regulator of cellulase production in T. reesei. We applied two powerful genomics methods (transcriptome sequencing and chromatin accessibility sequencing) to unravel the global role of Nsd3 and its regulatory mechanism. Nsd3 participates in various biological processes in T. reesei, including cell wall remodeling, calcium metabolism, and secondary metabolism, in addition to regulating the expression of sugar transporters. Protein-DNA interaction assays demonstrate that Nsd3 acts through important genes to regulate cellulase expression, including ace4, crt1, stp1, and cel1b. Our study provides mechanistic insights about how Nsd3 regulates diverse physiological processes in T. reesei. This work also applied ATAC-Seq for the first time to study chromatin accessibility in T. reesei.

ATAC-Seq↗

PubMed1994

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

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.

Bacterial Proteins↗

PubMed2003

Xylanase production by Trichoderma reesei rut C-30 on rice straw.

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.

Cellulase↗

PubMed1987

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

Amidohydrolases↗