SEARCH · PubMed Health

Results for “Trichoderma reesei”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed1999

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

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 micromol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 micromol 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.

Journal Article↗

PubMed2002

Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei.

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

Base Sequence↗

PubMed1999

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei.

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

Carbohydrate Sequence↗

PubMed2001

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Base Sequence↗

PubMed2002

Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing.

Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic in hibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley.

Amino Acid Sequence↗

PubMed1991

Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccharomyces cerevisiae.

The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3'-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADC1 expression cassette. EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the The-Ser-rich hinge region and the conserved 'tail' are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.

Blotting, Western↗

PubMed2000

Adsorption of Trichoderma reesei CBHI cellulase on silanized silica.

Adsorption kinetics and surfactant-mediated elution of Trichoderma reesei CBHI cellulase were recorded in situ, at hydrophobic, silanized silica. Experiments were performed at different solution concentrations, ranging from 0.001 to 0.98 mg/mL. Adsorbed enzyme was partially elutable upon rinsing, with the amount of adsorbed mass remaining being highest at intermediate concentrations. In addition, the resistance to elution with buffer was generally lower at higher concentrations, and the resistance to elution with surfactant was generally lower at intermediate concentrations. These observations are tentatively explained with reference to a mechanism allowing for adsorption of associated monomers of CBHI as well as free monomers.

Adsorption↗

PubMed1990

The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain.

Six uridine auxotroph mutants of Trichoderma reesei QM 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as OMP-decarboxylase negative (pyr-) by a radiometric enzyme assay. Transformation to uridine prototrophy was achieved with the pyr4 gene of Neurospora crassa (up to 1500 transformants/micrograms) and with pyrA of Aspergillus niger (700-800 transformants/micrograms). In many transformants the PYR+ function seems to be present as extrachromosomal DNA. There is evidence for a correlation between the stability of transformants and integration of the vector in the genome whereas unstable transformants are obtained when autonomous replication of the plasmid occurs.

Blotting, Southern↗

PubMed1993

Characterization of the Trichoderma reesei cbh2 promoter.

A 613-bp fragment of the 5' upstream region of the Trichoderma reesei cbh2 gene (coding for the cellulolytic enzyme cellobiohydrolase II) has been isolated and sequenced. Fusion of this fragment to the E. coli uidA gene (coding for beta-glucuronidase) leads to--albeit low--expression of beta-glucuronidase activity in the presence of cellulose and upon the addition of low molecular weight inducers (sophorose, lactose) of cellobiohydrolase II. It also governed the formation of beta-glucuronidase activity during sporulation and its transport to the conidial surface. However, despite the presence of a signal peptide in the cbh2:uidA fusion, beta-glucuronidase was not secreted in T. reesei. Defined fragments of the 613-bp promoter region were isolated and used to identify areas involved in the regulation of cbh2 expression by protein-DNA binding assays. At least two binding areas--between -443/-363 and -363/-173, respectively--were identified. In both areas, the DNA-protein complex observed was appreciably larger when cell-free extracts from sophorose-induced mycelia were used. This suggests that at least one of the proteins regulating cbh2 transcription is itself induced by cellulose.

Base Sequence↗

PubMed1993

Crystallization and preliminary X-ray studies on the core proteins of cellobiohydrolase I and endoglucanase I from Trichoderma reesei.

The catalytic core domains of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma reesei have been crystallized using the hanging drop vapour diffusion method. In the case of CBHI, use of polyethylene glycol 20,000, and calcium chloride at low pH produced good quality single crystals suitable for X-ray studies. The crystals belong to a primitive orthorhombic space group with unit cell dimensions a = 84.0 A, b = 86.2 A, c = 111.8 A, and diffract beyond 2.0 A resolution. Bipyramidal crystals of EGI core were grown from ammonium sulphate at pH 7.5. The crystals are tetragonal, either P4(1)22 or the enantiomorph P4(3)22, with cell dimensions a = b = 101.8 A and c = 198.0 A, and at best diffract to a resolution of 2.5 A.

Ammonium Sulfate↗

PubMed1996

Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei.

The three-dimensional structures of endo-1,4-xylanase II (XYNII) from Trichoderma reesei complexed with 4,5-epoxypentyl beta-D-xyloside (X-O-C5),3,4-epoxybutyl beta-D-xyloside (X-O-C4), and 2,3-epoxypropyl beta-D-xyloside (X-O-C3) were determined by X-ray crystallography. High-resolution measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.

Benzoates↗

PubMed1998

Modes of action on cotton and bacterial cellulose of a homologous endoglucanase-exoglucanase pair from Trichoderma reesei.

The endoglucanase I (EGI) and the cellobiohydrolase I (CBHI) of the filamentous fungus Trichoderma reesei form a homologous pair of cellulolytic enzymes which nevertheless have different modes of action. We show here that the action of CBHI on bacterial microcrystalline cellulose results in efficient solubilisation but only a slow decrease in its degree of polymerisation. In contrast, the action of EGI results in a rapid decrease of the degree of polymerisation but less efficient overall solubilisation of the substrate. CBHI alone was practically inactive toward cotton which has a high initial degree of polymerisation and a complex morphology. EGI rapidly reduced the degree of polymerisation of cotton, and slowly solubilised part of it. Working synergistically, EGI and CBHI solubilised cotton more rapidly and to a greater extent than EGI alone. Our data are consistent with the exoglucanase nature of CBHI and also provide some evidence supporting its processive mode of action.

Bacteria↗

PubMed1990

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride.

The p-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HCl) that did not affect the tryptophan fluorescence of this enzyme. The Km of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combined reversibly with 1 molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.

Binding Sites↗

PubMed2001

Adsorption of Thermomonospora fusca E5 and Trichoderma reesei cellobiohydrolase I cellulases on synthetic surfaces.

The interfacial behavior of Thermomonosporafusca E5 and Trichoderma reesei cellobiohydrolase I (CBHI) cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that could be analyzed by solution-phase techniques. Circular dichroism spectroscopy of each cellulase, alone as well as in suspension with silica, was used to determine whether structural changes occurred on adsorption. Changes in spectra were observed for CBHI, but not for E5. Gel-permeation chromatography of the cellulase-particle suspensions showed that neither cellulase binds to silica, suggesting that changes in spectra for CBHI were a result of solution-phase phenomena. Microfiltration of cellulase-polystyrene suspensions showed that both cellulases bind to polystyrene. However, circular dichroism experiments with polysterene proved unworkable, owing to excessive light absorption by the polystyrene. Adsorption kinetics of each cellulase were recorded, in situ, at hydrophilic and silanized, hydrophobic silica surfaces using ellipsometry. Ellipsometric data recorded for each cellulase at hydrophilic silica showed insignificant adsorption. Binding did occur between each cellulase and silanized silica, most likely mediated through hydrophobic associations. Adsorption in this case was irreversible to dilution.

Actinomycetales↗

PubMed1993

Mode of action of endoglucanase III from Trichoderma reesei.

Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4'-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarrón and Claeyssens (1991) Cellulose '91, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Béguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.

Bacterial Proteins↗

PubMed1996

Two regulatory regions controlling basal and cellulose-induced expression of the gene encoding cellobiohydrolase I of Trichoderma reesei are adjacent to its TATA box.

The cellulolytic system of the filamentous fungus Trichoderma reesei is transcriptionally induced in the presence of the insoluble polymer cellulose. Previous studies have demonstrated that induction of the cellulose transcripts by cellulose requires basal expression of its own genes. To understand how basal expression controls cellulose-induced transcription of those genes, we analyzed the 5'-flanking region of the gene encoding cellobiohydrolase I (cbh1), the major member of the cellulase system, for the cis-acting region that is responsible for regulating basal and cellulose-stimulated expression. Using the promoter deletion approach and an appropriate reporter gene, the cis-acting region responsible for cellulose-stimulated transcription was localized between -241 and -72 bp relative to the TATA box. Deletion of this sequence did not affect the basal expression of the promoter, whereas deletion of 72 bp adjacent to the TATA box abolished basal expression of the cbh1 promoter. We therefore concluded that the cbh1 promoter is composed of two regulatory regions-one controls cellulose-induced transcription and the other is required for its basal expression.

Base Sequence↗

PubMed1987

Purification and characterization of a beta-glucosidase from Trichoderma reesei.

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.

Amino Acids↗

PubMed1985

Subcellular fractionation of a hypercellulolytic mutant, Trichoderma reesei Rut-C30: localization of endoglucanase in microsomal fraction.

The growing mycelia of Trichoderma reesei Rut-C30 are richly endowed with endoplasmic reticula and a variety of pleomorphic subcellular bodies. Mycelia of the culture growing in presence of avicel pH101 was fractionated in sucrose density gradients, and several morphologically and biochemically distinct fractions were isolated. Mycelia were homogenized in a Bead Beater, and the homogenate was freed of nucleus and wall fragments by low-speed centrifugation before fractionation. Organelle-free cytosol, which did not penetrate the gradient, contained (of the total) 72% of the vanadate-sensitive ATPase, 26% of carboxymethyl cellulase (CMCase), 2% of cytochrome c reductase, and 13% of the protein. Significant fractions separated on a gradient were light vesicles containing heavily stained material inside and ribosomes attached to the outside surface, intact vesicles resembling condensing vacuoles, large vesicles derived from the plasma membrane, and heavy vesicles containing crystalline material. The light-vesicle fraction contained a large portion of the cell-bound CMCase activity. The particle-bound ATPase and cytochrome c reductase activities were concentrated in heavy fractions. The fractionation in the presence of MgCl2 improved the preservation of subcellular bodies derived from the endoplasmic reticula. Although the CMCase activity of the light-vesicle fraction was 4 times higher than the activity in the heavy-vesicle fraction, the CMCase antibody-binding capacities of both fractions were about the same. This discrepancy between the catalytic activity and the antibody-binding capacity suggests that the heavy vesicles might have contained considerable amount of inactive CMCase compared with that present in the light vesicles.

Cell Fractionation↗