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 199 records · Page 11Linked to original sources

PubMed1994

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

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.

Amino Acid Sequence↗

PubMed1996

Effect of cassavar cultivar, age and pretreatment processes of cellulase and xylanase production from cassava waste by Trichoderma harzianum.

Cassava wastes--the peel and the root fibre were taken through various pretreatment procedures before being subjected to solid state fermentation with Trichoderma harzianum. Most of the pretreatment processes increased the cellulose and hemicellulose content of the cassava peel and fibre by as high as 155% while sulfuric acid treatment resulted in 25.3% loss in the peel hemicellulose. The best pretreatment found to be 1% NaOH at 120 degrees C gave the highest production of the Cx, the Cl and xylanase enzymes with the cassava root fibre. Xylanase and cellulase production with the exception of the Cx was found to be affected by age while an improved cassava variety TMS(2) 1425 peel and fibre rated highest in terms of production of the enzymes. Percentage hydrolysis within range of 56.52-67.64% were recorded for the enzymes on sorghum grains.

Age Factors↗

PubMed1995

Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.

Adsorption↗

PubMed1997

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.

Cellulase↗

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↗

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↗

PubMed1995

Inhibition of cellobiohydrolase I from Trichoderma reesei by palladium.

Cellulase from Trichoderma reesei is a multienzyme mixture that hydrolyzes cellulose to glucose. Two enzymes in this mixture, cellobiohydrolase (CBH) and endoglucanase (EG), possess a common structure comprising a distinct cellulose-binding domain (CBD) and catalytic domain. Inhibition of the catalytic domain of cellulases without affecting their CBD function might be useful for structure/function studies of these enzymes. Complexes of the platinum group metals were tested for their ability to inhibit the major cellulase enzyme from T. reesei, cellobiohydrolase I (CBH I). Only palladium complexes inhibited CBH I, inhibition being dependent upon the molar ratio of palladium to CBH I with 1 microM CBH I retaining only 10% of its activity in the presence of 100 microM ammonium hexachloropalladate(IV) and after the incorporation of 28 mol Pd/mol CBH I. Inhibition was irreversible and could be completely prevented by including histidine, cysteine, and cystine in the assay mixture. Although the primary mechanism of inhibition of CBH I by palladium remains to be elucidated, it could involve the binding of palladium to sulfur or cystine residues resulting in their degradation. This is based on the findings that (i) palladium-inhibited CBH I was less thermally stable than native CBH I; (ii) CBH I, chemically modified by the attachment of pentaammine ruthenium(III) to the imidazole-N of either H206 or H228, showed greater sensitivity to inhibition by palladium compared to native CBH I; and (iii) ammonium hexachloropalladate cleaved 5,5'-dithiobis(2-nitrobenzoic acid)--Ellman's reagent. Binding of CBH I to crystalline cotton linters was not affected by palladium.

Amino Acids↗

PubMed2001

Enzymatic synthesis of aliphatic beta-lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase.

Aliphatic beta-lactosides were directly synthesized by beta-lactosyl transfer reaction from p-nitrophenyl beta-lactoside (Lac beta-pNP) to various 1-alkanols (n = 2-12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl beta-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl beta-lactoside (2) and dodecyl beta-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds (5-13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl beta-N-acetyllactosaminide (LacNAc beta-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Gal beta1-4GlcNAc beta1-4Man (4) and Gal beta1-4GlcNAc beta1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE-Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.

Amino Sugars↗

PubMed1993

Attachments of pentaammineruthenium(III) to Trichoderma reesei cellobiohydrolase I increases its catalytic activity.

Pentaammineurthenium(III) was covalently attached to cellobiohydrolase I (CBH I, EC 3.2.1.91), the major component of Trichoderma reesei cellulase, resulting in 0.7 mol ruthenium/mol CBH I and an electrode potential of +95 mV. Fractionation of modified CBH I by chromatofocusing resulted in the separation of fractions with a 1.4- to 3.2-fold increase in specific activity toward p-nitrophenylcellobioside, depending on the assay conditions, over that of native enzyme. The extent of the hydrolysis of insoluble cellulosic substrates (Avicel and newsprint) to glucose by modified CBH I was also greater than that observed by the native enzyme.

Cellulose 1,4-beta-Cellobiosidase↗

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↗

PubMed1998

alpha-Mannosidase from Trichoderma reesei participates in the postsecretory deglycosylation of glycoproteins.

The 160 kDa alpha-mannosidase (E.C. 3.2.1.24) isolated from culture filtrate of Trichoderma reesei has wide aglycon specificity but cleaves the alpha1 --> 2 and alpha1 --> 3 mannosidic bonds with higher rate than alpha1 --> 6 bond and slowly hydrolyses yeast mannan and 1,6-alpha-mannan. The specific activity of the enzyme and rate constant in the reaction with p-nitrophenyl-alpha-D-mannopyranoside were 0.15 U/mg and 1.62 x 10(-4) microM/min/microg, respectively, at optimal pH 6.5. We have found that in vitro enzyme is able to cleave off 30% of total alpha-mannopyranosyl residues from N- and O-linked glycans of secreted glycoproteins. The activity of the alpha-mannosidase toward glycoproteins in vivo was studied comparing the structures of O- and N-linked glycans of glycoproteins isolated from the cultures growing with and without 1-deoxymannojirimycin, an inhibitor of alpha-mannosidases. Difference in structures of these glycans may be explained by postsecretory deglycosylation catalysed by the alpha-mannosidase.

1-Deoxynojirimycin↗

PubMed1998

A region of the cellobiohydrolase I promoter from the filamentous fungus Trichoderma reesei mediates glucose repression in Saccharomyces cerevisiae, dependent on mitochondrial activity.

The upstream activating region that controls cellulose-induced expression of the glucose-repressible cellobiohydrolase I gene (UARcb1) of the filamentous fungus Trichoderma reesei is shown to mediate transcription and glucose repression of a reporter gene in Saccharomyces cerevisiae, a unicellular microorganism that lacks the genes required for the utilization of cellulose. Glucose-controlled transcription mediated by UARcb1 requires the products of the genes SNF1 and SSN6, a protein kinase and a repressor, respectively, that regulate glucose-repressible yeast genes. Previously, it has been shown that mitochondrial function is implicated in cellobiohydrolase I gene expression in T. reesei and this sensitivity to the metabolic state of the mitochondria was shown to be transcriptionally controlled by the 5'-flanking sequence of the cbh1 gene [Abrahão-Neto et al. (1995) Biochemistry 34, 10456-10462]. Remarkably, transcription of the reporter gene controlled by UARcb1 in S. cerevisiae also showed a requirement for active mitochondria, suggesting that a common mechanism involving mitochondrial activity controls glucose-repressible genes in both microorganisms.

5' Untranslated Regions↗

PubMed1998

The arg2 gene of Trichoderma virens: cloning and development of a homologous transformation system.

The arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.

Amino Acid Sequence↗

PubMed1999

Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host.

For monitoring chitinase expression during mycoparasitism of Trichoderma harzianum in situ, we constructed strains containing fusions of green fluorescent protein (GFP) to the 5'-regulatory sequences of the T. harzianum nag1 (N-acetyl-beta-d-glucosaminidase-encoding) and ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. harzianum onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. harzianum. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.

Acetylglucosaminidase↗

PubMed1999

Developmental regulation of cmp1, a gene encoding a multidomain conidiospore surface protein of Trichoderma.

A gene encoding a developmentally regulated polypeptide of Trichoderma (strain ATCC 32173) was isolated, with the help of an antibody against a 62-kDa protein whose abundance strongly increases during photoinduced sporulation. The amino acid sequence deduced from this gene, cmp1 (conidial multidomain protein), is a 135-kDa polypeptide consisting of several domains. Although reminiscent of known structural modules, two of the domains may define novel families. The protein is apparently processed to give the 62-kDa species. Immunogold labeling electron microscopy localized the antigen to the membrane or inner wall layers. The mRNA is strongly up-regulated during sporulation. At least part of this regulation is likely to be conferred by several elements identified in the upstream region, with homology to elements recognized by fungal transcription factors for regulation by conidiation, light, and nitrogen stress. The developmental regulation, cell surface location, and modular structure suggest a function in cell-cell interactions, detection of the wall by the cell, or anchoring of the plasma membrane to the wall.

Amino Acid Sequence↗

PubMed1993

Crystallization of alpha-galactosidase from Trichoderma reesei.

An extracellular alpha-galactosidase was isolated from fungus Trichoderma reesei. The purified enzyme had a molecular weight of 54,000 Da and an isoelectric point of 5.25. Crystals of alpha-galactosidase were obtained from a polyethylene glycol 4000 solution by the hanging-drop method. Seeding was used for enlargement of the crystal size. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 122.2 A, b = 81.0 A c = 49.7 A and diffract beyond 3.0 A resolution.

Crystallization↗

PubMed1993

Crystallization and preliminary X-ray analysis of two major xylanases from Trichoderma reesei.

Two major endoxylanases, endo-beta-1,4-xylanase I and II (molecular mass 19 kDa and 21 kDa) from the filamentous fungus Trichoderma reesei have been crystallized by using ammonium sulphate as the precipitating agent. Both crystals were monoclinic and belonged to the space groups C2 (a = 71.9 A, b = 39.0 A, c = 59.9 A, beta = 118.0 degrees, for XYNI) and P2(1) (a = 81.6 A, b = 60.6 A, c = 38.3 A, beta = 94.4, for XYNII). The crystals diffract to at least 2.2 A and 1.5 A, respectively.

Crystallization↗

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↗