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PubMed1997

cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast.

A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulose-binding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporus cellulose-induced protein CEL1 whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene egl4. Polyclonal antibodies were prepared against EGIV produced in Escherichia coli and detected a 56-kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus.

Agaricus↗

PubMed1997

Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30.

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the alpha-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The alpha-1,6 arm shows a remarkable heterogeneity: in addition to alpha-1,2-Man and alpha-1,6-Man, the presence of a terminal mannose alpha-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant N-glycans on cellobiohydrolase I can be represented as follows: GlcMan8GlcNAc2, GlcMan7GlcNAc2, Man7GlcNAc2, ManPGlcMan7GlcNAc2, GlcMan5GlcNAc2 and Man5GlcNAc2.

Carbohydrate Sequence↗

PubMed1997

In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides--evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei.

To investigate the potential of filamentous fungi to synthesize N-glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N-acetylglucosaminyltransferase I, human beta-1,4-galactosyltransferase and rat alpha-2,6-sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High-mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian-type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoi alpha-1,2-mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N-glycan processing in T. reesei apparently involves different steps, including alpha-1,2-mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The alpha-1,2-mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.

Acetylglucosamine↗

PubMed2004

Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.

Alkaline Phosphatase↗

PubMedDate not supplied

Trichoderma viride infection in a liver transplant recipient.

A liver transplant recipient developed infection of a perihepatic haematoma due to Trichoderma viride. Before the infection was diagnosed, the patient received intense immuno-suppressive and prolonged antibacterial and anti-fungal therapies. Although the death of the patient was not directly related to the fungal infection, abundant quantities of this pathogen were recovered in the haematoma at post-mortem examination, despite previous surgical removal and treatment with amphotericin B and fluconazole.

Adult↗

PubMed1987

beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases.

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.

Aspergillus↗

PubMed1995

Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei.

The pH optimum, temperature-dependence, thermal stability, substrate specificity and subsite affinities of the 66 kDa, pI 4.0 glucoamylase of the filamentous fungus Trichoderma reesei were determined. It had a pH optimum of 5.5 and a temperature optimum (5 min reaction time) of 70 degrees C with soluble starch as substrate. Thermal-inactivation studies revealed that the glucoamylase is relatively thermostable up to 60 degrees C. Metal ions and EDTA tested at 5 mM concentrations had no significant effect, and beta-cyclodextrin only slightly inhibitory effects, on the digestion of soluble starch. Estimated Km and kcat. values for soluble starch where 0.11 mg.ml-1 and 28.5 s-1 respectively. Hydrolysis of pullulan (Km 14 mg.ml-1 and kcat. = 6.6 s-1) indicated substantial activity towards 1,6-O-glucosidic bonds. From ratios of kinetic parameters of malto- and isomalto-oligosaccharides, it was apparent that the glucoamylase showed approx. 3-fold higher selectivity towards isomalto-oligosaccharides than most other reported fungal glucoamylases. Substrate binding affinities were calculated from kinetic data for the linear series of malto- and isomalto-oligosaccharides. The results were in good agreement with other reported glucoamylases. The main difference was that subsite 1 showed a slightly negative free energy of binding with malto-oligosaccharides, whereas most other glucoamylases show a positive free energy at this subsite. A set of peptides obtained from purified glucoamylase by tryptic digestion where sequenced. They covered approx. 17% of the total amino acid sequence as estimated from molecular mass on SDS/PAGE. Some of the sequences were tentatively aligned to known glucoamylase sequences. They showed about 60% identity with the extensively studied Aspergillus glucoamylase.

Amino Acid Sequence↗

PubMed1998

Action of Trichoderma reesei and Aspergillus oryzae esterases in the deacetylation of hemicelluloses.

Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The functions of hemicellulose-deacetylating esterases of Trichoderma reesei and Aspergillus oryzae are discussed in this paper. Both fungi produce multiple esterases and two different esterases were isolated from both T. reesei and A. oryzae. The enzymes differed significantly in their substrate specificities. Acetyl xylan esterase of T. reesei was highly active on polymeric xylan but was unable to remove acetyl substituents from glucomannan or phenolic substituents from wheat straw arabinoxylan. Another esterase, acetyl esterase from T. reesei, had activity only towards short oligomeric and monomeric acetates derived both from xylan and glucomannan. The acetyl glucomannan esterase of A. oryzae was most active towards polymeric glucomannan, but was also able to remove acetyl groups from xylan. The only esterase studied which was active against phenolic substituents in arabinoxylans was the feruloyl esterase from A. oryzae. Feruloyl esterase had the widest substrate specificity of the esterases studied. It was also able to act on acetyl groups both in xylan and in glucomannan. The simultaneous enzymic liberation of acetyl groups from xylan and glucomannan clearly enhanced the action of xylan- and mannan-degrading enzymes, thus increasing the hydrolysis yield significantly. However, none of the esterases was able to remove all acetyl substituents when acting alone and simultaneous action of two esterases was needed for complete deacetylation.

Acetates↗

PubMed2004

Construction of a yeast one-hybrid system with the xylanase2 promoter from Trichoderma reesei to isolate transcriptional activators.

AIMS: To construct a yeast one-hybrid system and isolate transcriptional activators. METHODS AND RESULTS: A 1.1-kb promoter region of xylanase2 from Trichoderma reesei was cloned by PCR and sequenced (GenBank accession number: AY263380). Sequence analysis revealed that typical binding sites for several transcription factors in filamentous fungi, such as CREI, XLNR, ALCR, AREA and CCAAT enhancer, are located in the promoter. To isolate xyn2 transcription factors, the reporter plasmid of a yeast one-hybrid system was constructed on the backbone of the plasmid pRS415 containing the leu2 selective marker, with the xyn2 promoter region and Saccharomyces cerevisiae his4 as a reporter gene. The reporter gene contained 123-bp minimal promoter region. The S. cerevisiae H158 strain containing the reporter plasmid was transformed with a T. reesei expression cDNA library, and 34 transformants were collected from SC-Leu-His-Ura plates. The isolation of the gene ace2 from several transformants showed that the one-hybrid system approach was successful. Then, approx. 59 mg l(-1) of ace2 was overexpressed in Escherichia coli BL21. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast one-hybrid system is suitable for isolating transcription factors of filamentous fungi. ACE II is a main and universal transcriptional activator that controls cellulase and hemicellulase transcription regulation in T. reesei.

Alcohol Oxidoreductases↗

PubMed2006

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens.

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.

4-Butyrolactone↗

PubMed2006

A microsatellite based method for quantification of fungi in decomposing plant material elucidates the role of Fusarium graminearum DON production in the saprophytic competition with Trichoderma atroviride in maize tissue microcosms.

Common PCR assays for quantification of fungi in living plants cannot be used to study saprophytic colonization of fungi because plant decomposition releases PCR-inhibiting substances and saprophytes degrade the plant DNA which could serve as internal standard. The microsatellite PCR assays presented here overcome these problems by spiking samples prior to DNA extraction with mycelium of a reference strain. PCR with fluorescent primers co-amplifies microsatellite fragments of different length from target and reference strains. These fragments were separated in a capillary sequencer with fluorescence detection. The target/reference ratio of fluorescence signal was used to calculate target biomass in the sample. Such PCR assays were developed for the mycotoxin deoxynivalenol (DON)-producing wheat and maize pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride, using new microsatellite markers. In contrast to real-time PCR assays, the novel PCR assays showed reliable fungal biomass quantification in samples with differentially decomposed plant tissue. The PCR assays were used to quantify the two fungi after competitive colonization of autoclaved maize leaf tissue in microcosms. Using a DON-producing F. graminearum wild-type strain and its nontoxigenic mutant we found no evidence for a role of DON production in F. graminearum defense against T. atroviride. The presence of T. atroviride resulted in a 36% lower wild-type DON production per biomass.

Antibiosis↗

PubMed1990

Characterization of an unglycosylated low molecular weight 1,4-beta-glucan-glucanohydrolase of Trichoderma reesei.

A low molecular weight endoglucanase (1,4-beta-glucan glucanohydrolase E.C.3.2.1.4) was purified to homogeneity by a two-step procedure from 7 day old culture filtrates of Trichoderma reesei. The endoglucanase was obtained by BioGel A 0.5 m gel chromatography followed by preparative PAGIF. The purified endoglucanase was homogeneous upon titration curve separation. Enzyme characteristics were: Mr 25 kDa, pI 7.5. The amino acid composition is predominantly neutral (mainly glycine). The N-terminus is arginine. The pH-optimum for this endoglucanase was 5.8 and its optimal temperature was at 52 degrees C. The activity of this endoglucanase gave a strong increase in CMC-fluidity with only a small release of reducing sugars. The endoglucanase was 0.2% of total culture medium protein content. The reducing sugars upon CMC digestion were G1-G4. The enzyme had no specificity towards crystalline cellulose (Avicel) or xylan. The endoglucanase is not a glycoprotein.

Amino Acids↗

PubMed1993

Formation of cross-fractures in cellulose microfibril structure by an endoglucanase-cellobiohydrolase complex from Trichoderma reesei.

An endoglucanase-cellobiohydrolase complex from Trichoderma reesei culture fluids was purified by means of preparative isoelectric focusing. The cellulase complex had a common apparent isoelectric point (pI) of 3.8. Beyond this pI, the electrophoretic mobilities of endoglucanase and cellobiohydrolase were different under conditions of titration curves. The effect of this endoglucanase-cellobiohydrolase complex on Sinapis cellulose microfibril ultrastructure was observed by transmission electron microscopy after metal shadowing of the specimen. By the action of this cellulase complex, the microfibril structure was converted into an amorphous form of cellulose. Moreover, the hydrolase complex induced visible cross-fractures within the cellulose microfibril structure. The mean cellulose microfibril length of 1.2 microns was reduced to 0.9 microns in the presence (12 h) of this cellulase complex by the formation of shorter microfibril fragments.

Biodegradation, Environmental↗

PubMed1993

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal.

Secretion of the Hormoconis resinae glucoamylase P (GAMP) enzyme from Trichoderma reesei using either the natural N-terminal extension of the premature glucoamylase P or the cellobiohydrolase I (CBHI) signal peptide was examined. The expression conditions for the heterologous glucoamylase P (gamP) gene in T. reesei were standardized by targeting one copy of a plasmid fragment, containing the gamP gene, to the cbh1 locus of the host. The results showed that the transient N-terminal extension of the premature GAMP acts as an efficient secretion signal in T. reesei and leads to a higher yield of extracellular glucoamylase activity than does the signal peptide of CBHI.

Amino Acid Sequence↗

PubMed1993

Raffinose-induced mutanase production from Trichoderma harzianum.

The enzyme alpha(1-->3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15,000 M(r) protein that was also regulated by carbon source and by illumination conditions.

Enzyme Induction↗

PubMed1994

Transcriptional regulation of the Trichoderma longibrachiatum egl1 gene.

Transcription of the Trichoderma longibrachiatum egl1 gene is induced in the presence of lactose and beta-methylglucoside and repressed by glucose. A DNA fragment containing 722 bp upstream of the ATG codon has been sequenced. The gene has two major transcription start points (20 and 24 nucleotides upstream from the ATG codon) and several transcription termination points (located in a region around 130 nt downstream of the stop codon). Two 6-mer sequences (5'-CTGGAG-3') separated by 16 bp are present in the egl1 gene promoter. These sequences match the Aspergillus nidulans consensus CreA binding site and might be implicated in carbon catabolite repression of egl1 transcription.

Base Sequence↗

PubMed1996

Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region.

A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene (cbh1) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein::Cre1(10-131) fusion protein (Cre1 is a catabolite repressor of T. reesei) and the cbh1 upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position -1496 to -993) bearing three 6-bp sites, A1, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position -994 to -639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.

Base Sequence↗

PubMed2000

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum.

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.

Chitin↗