SEARCH · PubMed Health

Results for “Trichoderma”

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 109 records · Page 6Linked to original sources

PubMed1986

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Amino Acid Sequence↗

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↗

PubMed1988

The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei.

We have identified a genomic DNA fragment from the lignin-degrading fungus Phanerochaete chrysosporium (P.c.) that hybridizes to a DNA probe encoding part of the exo-cellobiohydrolase I (CBHI) gene of Trichoderma reesei (T.r.). This fragment has been subcloned and its nucleotide sequence determined. We demonstrate that it could encode a 516 residue protein that shows strong homology with the known protein sequence of CBHI from T.r. Comparison of the two nucleotide sequences identifies two regions within the P.c. sequence that are not represented in T.r. Further inspection of these regions reveals sequences closely related to the conserved elements of filamentous fungal introns. We conclude that the P.c. genomic sequence contains the same number of introns (2) as found in T.r. but that these are located at different relative positions within the two genes. The transcript from the P.c. sequence is induced in the presence of cellulose but not by glucose and we therefore conclude that this sequence represents the first cellulase gene to have been described from this organism.

Amino Acid Sequence↗

PubMed1988

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

Amino Acid Sequence↗

PubMed1989

A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei.

The nucleotide sequence of a cDNA coding for a lignin peroxidase (Lgp) of the white-rot fungus, Phlebia radiata, has been determined. By amino acid (aa) sequencing, it has been shown that the protein product of this gene is the LIII Lgp of Pb. radiata. The isolated gene and the putative aa sequence are about 60% homologous to published Lgp sequences from the fungus, Phanerochaete chrysosporium. The aa thought to be involved in the catalysis of LIII are revealed by comparison with the yeast cytochrome c peroxidase. The P. radiata Lgp-encoding gene (lgp3) was expressed in the fungus, Trichoderma reesei, under the cellobiohydrolase-encoding cbh1 gene promoter. Lgp3 mRNA was produced by the T. reesei transformants. No Lgp protein, however, could be detected.

Agaricales↗

PubMed1991

Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene (pgk1) of Trichoderma reesei.

Transcription of the 3-phosphoglycerate kinase (PGK)-encoding gene (pgk1) of Trichoderma reesei results in two transcripts due to two main transcription start points (tsp) which are differentially regulated during the growth cycle. The nucleotide sequence of the promoter reveals a number of putative regulatory elements present also in the PGK promoter of Saccharomyces cerevisiae: a 20-nt long sequence similar to the CTTCC-repeat region of the upstream activating sequence UAS, the eukaryotic heat-shock consensus sequence, HSE, and a putative eukaryotic cAMP regulatory sequence. The functionality of the putative HSE sequence was examined, but no clear effect could be seen on the total amount of pgk1 mRNA at elevated temperatures nor on transcription initiation from the upstream tsp, preceded by the HSE sequence.

Amino Acid Sequence↗

PubMed1979

Preparation and properties of pectic enzymes produced by Trichoderma lignorum.

Polygalacturonase and protein-methylesterase were isolated from shaken culture of Trichoderma lignorium. Isolation was carried out with various agents. Methanol was the most suitable precipitant for isolating polygalacturonase, yielding enzyme preparations 6.6 times more active than that of culture filtrate. Likewise, tannin afforded active fractions at pH 4 and 0.05% concentrations. Similarly, 50% ammonium sulphate saturation gave active fractions. The least polygalacturonase activity was obtained from ethanol. In any of the organic solvents used, highest enzymic activity was obtained when using only one volume. As regards pectin-methylesterase, no correlation existed between its activity and concentration of the precipitant used. A substrate concentration above 0.8% was a limiting factor for polygalacturonase activity, while optimum enzyme concentration was 40 microgram protein/ml at 40 degrees C and pH 4.45.

Esterases↗

PubMed1989

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

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↗

PubMed1978

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.

Cellulase↗

PubMed1979

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.

Amino Acids↗

PubMed1985

A new appraisal of the endoglucanases of the fungus Trichoderma reesei.

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble beta-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.

Cellulase↗

PubMed1989

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

Cellulose 1,4-beta-Cellobiosidase↗

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↗

PubMed1991

Purification and characterization of an endoxylanase from Trichoderma koningii G-39.

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

Amino Acids↗

PubMed1992

Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414.

The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.

Ammonium Sulfate↗

PubMed1989

Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.

Cellulose↗