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PubMed1995

Cre1, the carbon catabolite repressor protein from Trichoderma reesei.

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Amino Acid Sequence↗

PubMed1992

The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes.

As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, we have purified two endo-beta-1,4-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and cloned their genes. The enzymes were isolated from culture filtrates of T. reesei C30 grown on xylan as a carbon source, using two steps of cation exchange chromatography. They exhibited molecular weights of 19 (XYN I) and 21 (XYN II) kD, and isoelectric points of 5.2 and 9.0, respectively. These enzymes differed in their pH optimum for activity and affinity for xylan, and accounted for more than 90% of the total xylanolytic activity of the fungus. The purified enzymes were subjected to N-terminal sequence analysis, and after cleavage with trypsin and endoproteinase Glu-C the resulting peptides were sequenced. Oligonucleotides based on these sequences were used to clone gene fragments via PCR, and these were used as probes to isolate full-length copies of xyn1 and xyn2 from a lambda gene bank of T. reesei. The products of xyn1 and xyn2 share considerable homology, but the enzyme encoded by xyn2 appears to more closely resemble several other bacterial and fungal xylanases than does that of xyn1.

Amino Acid Sequence↗

PubMed2005

Production of cellulase/beta-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure.

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH2PO4, 2 mL/L of Tween-80, and 2mg/L of CoCl2. However, beta-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the beta-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and beta-glucosidase (0.64 IU/mL). The ratio of beta-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p < 0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.

Animals↗

PubMed2005

Increased alkali stability in Trichoderma reesei endo-1, 4-beta-xylanase II by site directed mutagenesis.

A number of engineered Trichoderma reesei endo-beta-1,4-xylanase (Xyn II) mutants were created and activity tests were performed for increased stability. The stability of the earlier characterized mutant Y5 (T2C, T28C, K58R, +191D) was further increased by the mutations creating the constructs P9 (N97R+F93W+H144K), P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C). The resistance towards thermal inactivation at alkaline pH was increased in all of the mutants. Residual activity T(50%) was increased 4-5 degrees C for P9 at pH 9. The performance of the P9 mutant in sulphate pulp bleaching was also tested and was shown to increase brightness markedly compared to the reference. The bleaching results showed the industrial potential of the obtained mutant.

Alkalies↗

PubMed1998

Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties.

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Aspergillus oryzae↗

PubMed1998

Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei.

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.

Acetylglucosamine↗

PubMed1995

Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei.

Three-dimensional structures of two major endo-1,4-xylanases, XYNI and XYNII from Trichoderma reesei, have been determined by X-ray crystallography. The amino acid sequences of both enzymes are highly homologous (identity approximately 50%), and both XYNI and XYNII exist as a single domain that contains two mostly antiparallel beta-sheets which are packed against each other. The beta-sheet structure is twisted, forming a cleft where the active site is situated. Two glutamic acids in the cleft, Glu75 and Glu164 in XYNI as well as Glu86 and Glu177 in XYNII, are most likely involved in catalysis. Inspection of the structures reveals that the width of the active site cleft and the number of subsites are different in XYNI and XYNII. The active site is narrower in XYNI and probably contains only three subsites, whereas the number of subsites in XYNII is most likely five. Variations in the surroundings of catalytic residue Glu164XYNI/Glu177XYNII are thought to explain the pH optimum differences observed in XYNI and XYNII.

Amino Acid Sequence↗

PubMed1992

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

Adsorption↗

PubMed1990

Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I.

Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kcat., kcat./Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-O-beta-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of D-glucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The beta 1-4 configuration of the newly formed bond was proved.

Cellulase↗

PubMed1998

Two adjacent protein binding motifs in the cbh2 (cellobiohydrolase II-encoding) promoter of the fungus Hypocrea jecorina (Trichoderma reesei) cooperate in the induction by cellulose.

The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of several cellobiohydrolases, endoglucanases, and beta-glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disaccharide sophorose. Using cell-free extracts from sophorose-induced and noninduced mycelia and various fragments of the cbh2 promoter of H. jecorina in electrophoretic mobility shift assay (EMSA) analysis and performing in vitro and in vivo footprinting analysis, we detected the nucleotide sequence 5'-ATTGGGTAATA-3' (consequently named cbh2-activating element (CAE)) to bind a protein complex with different migration in EMSA of induced and noninduced cell-free extracts. EMSA analysis, employing oligonucleotide fragments containing specifically mutated versions of CAE, revealed that protein binding requires the presence of an intact copy of either one of two adjacent motifs: a CCAAT (=ATTGG) box on the template strand and a GTAATA box on the coding strand, whereas a simultaneous mutation in both completely abolished binding. H. jecorina transformants, containing correspondingly mutated versions of the cbh2 promoter fused to the Escherichia coli hph gene as a reporter, expressed hph in a manner paralleling the efficacy of CAE-protein complex formation in EMSA, suggesting that the presence of either of both motifs is required for induction of cbh2 gene transcription. Antibody supershift experiments with anti-HapC antiserum as well as EMSA competition experiments with CCAAT binding promoter fragments of the Aspergillus nidulans amdS promoter suggest that the H. jecorina CCAAT box binding complex contains a homologue of HapC. The nature of the adjacent, GTAATA-binding protein(s) and its cooperation with the HapC homologue in cbh2 gene induction is discussed.

Base Sequence↗

PubMedDate not supplied

Isolation of cellulolytic enzymes from Trichoderma reesei QM 9414.

Two cellobiohydrolases and two endoglucanases were purified from a culture filtrate of the fungus Trichoderma reesei QM 9414 by simple and straightforward purification techniques. Molecular weights, isoelectric points, amino acid compositions, and carbohydrate contents are reported.

Amino Acid Sequence↗

PubMed1999

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

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.

Amino Acid Sequence↗

PubMed1991

Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes.

Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (greater than 10(4) transformants per micrograms DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.

Amino Acid Sequence↗

PubMed1991

Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation.

We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5'-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.

Amino Acid Sequence↗

PubMed2005

New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei.

A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, beta-xylosidase, alpha-L-arabinofuranosidase, acetyl xylan esterase, mannanase, alpha-galactosidase, xyloglucanase, polygalacturonase, and exo-beta-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in "cellulase" and "xylanase" fermentation conditions are shown.

Binding Sites↗

PubMed2001

Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

Amino Acid Sequence↗

PubMed1983

Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry.

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.

Cellulase↗

PubMed2002

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric.

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.

Cellulase↗