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PubMed1997

Structural and functional properties of low molecular weight endo-1,4-beta-xylanases.

There are currently four crystal structures of low molecular weight endo-1,4-beta-xylanases (E.C.3.2.1.8), i.e. family G/11 xylanases, available at the Brookhaven Data Bank: 2 xylanases from Trichoderma reesei (Törrönen et al., 1994; Törrönen and Rouvinen, 1995) and one from Bacillus circulans and another from Trichoderma harzianum (Campbell et al., 1993). They consist of two beta-sheets and one alpha-helix and have been described to resemble a partly-closed right hand. The catalytic residues are two conserved glutamate residues, which are located opposite to each other in an open active site cleft. The catalytic mechanism is thought to resemble that of the widely-studied enzyme lysozyme. The role of one glutamate is to act as an acid/base catalyst whereas the other is a nucleophile and stabilizes the reaction intermediate. Complex structures of partly-bound xylotetraose in mutated XYN from Bacillus circulans (Wakarchuck et al., 1994a) and three recently-obtained structures of XYNII from Trichoderma reesei with epoxyalkyl-xylose derivatives (Havukainen et al., 1996) have provided important information on substrate binding. Family G/11 xylanases show clear amino acid homology and thus have a common fold. However, variations in their functional properties, such as catalytic activity, substrate cleaving patterns, pH optima and thermostabilities, exist.

Amino Acid Sequence↗

PubMed1998

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus.

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Amino Acid Sequence↗

PubMed2002

Filamentous fungi in Indah Water Konsortium (IWK) sewage treatment plant for biological treatment of domestic wastewater sludge.

A study was carried out to isolate and identify filamentous fungi for the treatment of domestic wastewater sludge by enhancing biodegradability, settleability and dewaterability of treated sludge using liquid state bioconversion process. A total of 70 strains of filamentous fungi were isolated from three different sources (wastewater, sewage sludge and leachate) of IWK's (Indah Water Konsortium) sewage treatment plant, Malaysia. The isolated strains were purified by conventional techniques and identified by microscopic examination. The strains isolated belonged to the genera of Penicillium, Aspergillus, Trichoderma, Spicaria and Hyaloflorae The distribution of observed isolated fungi were 41% in sewage sludge followed by 39% in wastewater and 20% in leachate. The predominant fungus was Penicillium (39 strains). The second and third most common isolates were Aspergillus (14 strains) and Trichoderma (12 strains). The other isolates were Spicaria (3 strains) and Hyaloflorae (2 strains). Three strains (WWZP1003, LZP3001, LZP3005) of Penicillium (P. corylophilum, P. waksmanii, and P. citrinum respectively), 2 strains (WWZA1006 and SS2017) of Aspergillus (A. terrues and A. flavus respectively) and one strain (SSZT2008) of Trichoderma (T. harzianum) were tentatively identified up to species level and finally verified by CABI Bioscience Identification Services, UK.

Biodegradation, Environmental↗

PubMed1987

Acifluorfen Enhancement of Cryptochrome-Modulated Sporulation following an Inductive Light Pulse.

Acifluorfen enhancement of blue-light mediated phototropism suggested that this diphenyl-ether herbicide augments the light reaction (TY Leong, WR Briggs 1983 Plant Physiol 70: 875-881). The separation of the possible direct interaction of acifluorfen with light reactions from interactions with dark pathways has been elucidated in this paper with Trichoderma harzianum. Acifluorfen at 30 micromolar, given for 5 hours in the growth medium, stimulated the conidiation of Trichoderma in response to blue light without apparently affecting growth. Enhanced conidiation could be elicited by dipping cultures into medium with acifluorfen both before as well as 0.5 hour after inductive blue light. This postphotoinduction stimulation indicates that acifluorfen does not directly augment the effect of light by interacting with cryptochrome(s) in Trichoderma. Instead, acifluorfen most probably interacted with the dark reactions following photoinduction.

Journal Article↗

PubMed2000

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus.

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.

Amino Acid Sequence↗

PubMed2004

Etest for assessing the susceptibility of filamentous fungi.

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.

Antifungal Agents↗

PubMedDate not supplied

[Fungal decomposition of oat straw during liquid and solid state fermentation].

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.

Antibiosis↗

PubMed2002

A protocol for evaluation of the role of disinfectants in limiting pathogens and weed moulds in commercial mushroom production.

In vitro and in vivo evaluations of the effects of three commercial disinfectants on isolates of Trichoderma harzianum Rifai and Cladobotryum dendroides (Bull) W Gams & Hoozemans were combined in the development of methodologies for realistic assessment of disinfectant performance. 'Environ' and 'Purogene' incorporated into agar media at 50 mg AI litre-1 were effective in totally preventing the mycelial growth of T harzianum and C dendroides isolates, whereas with 'Sudol' a concentration of 500 mg AI litre-1 was required. Evaluation of a model in vivo system was combined with observations on the fungicidal effects of disinfectants. Spore suspensions of T harzianum (Th1), T harzianum (Th2) and C dendroides prepared from culture washings with sterilised distilled water were used as contaminating inoculum. Environ, Sudol and Purogene in aqueous solutions at 1000 mg AI litre-1 were sprayed onto these building structure surfaces before or after artificial contamination with spore suspensions. Re-isolation from surfaces was carried out using agar swabs, which were prepared in visking tubing, applied to treated surfaces and incubated at 25 degrees C for 24, 48, 72 or 96 h. Environ and Purogene were more effective than Sudol in limiting the recovery of Trichoderma spp and C dendroides. All three disinfectants applied 12 h after artificial contamination onto wood, concrete or glazed surfaces were able to reduce the recovery of these isolates more effectively than when they were applied 12 h before artificial contamination. Greater persistence of contamination was noted on the rougher surfaces (wood and concrete) than on the smoother glazed surface. It was concluded in this in vivo evaluation that, in many cases, the protection achieved by the physical nature of the smooth glazed tile surfaces was equivalent to that available from the application of disinfectants, thus highlighting the attention needed to the nature of building surfaces in the structures used in the mushroom industry. The artificial contamination and re-isolation techniques allowed significant comparison between disinfectant materials, timings and surface types. In the in vivo experiments using a single standardised test concentration, the relative fungicidal effects of the disinfectants, which varied in the complexity of their active ingredients, were better discriminated than the effects observed in vitro, although the general trend was similar.

Agaricales↗

PubMed1998

Cysteine protease inhibitor from pearl millet: a new class of antifungal protein.

A cysteine protease inhibitor exhibiting antifungal activity from pearl millet seeds has been purified to homogeneity by ammonium sulphate precipitation and chromatographic procedures involving CM- sephadex and SP-sepharose cation exchange columns. The molecular characterization has revealed its molecular mass as 24 kD and isoelectric point 9.8. The amino acid composition data shows presence of high content of serine and glycine (34 residues/mole) and absence of tryptophan. The inhibitor exhibits potent antifungal activity against Trichoderma reesei, a dead wood fungus with minimum inhibitory dose to inhibit mycelial growth or spore germination is as low as 1 microgram/ml (250 ng/disc). In addition to Trichoderma reesei, the antifungal activity is observed against some important phytopathogenic fungi, namely, Claviceps, Helminthosporium, Curvularia, Alternaria and Fusarium species. To the best of our knowledge, a cysteine protease inhibitor as an antifungal protein is reported for the first time from a plant system.

Amino Acids↗

PubMed1993

DNA- and PCR-fingerprinting in fungi.

DNA-fingerprinting has been successfully used to detect hypervariable, repetitive DNA sequences (minisatellites and microsatellites) in fungi. Combined with methods used to identify random amplified polymorphic DNA (RAPD), conventional DNA-fingerprinting hybridization probes can also be used as single primers to detect DNA polymorphisms among fungal species and strains. The oligonucleotides (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, as well as the phage M13 and its core sequence, have been used as specific probes in hybridization experiments and as primers for PCR analysis. Both methods have enabled the differentiation of all the fungal species and strains that were examined, including species of Penicillium, Trichoderma, Leptosphaeria, Saccharomyces, Candida and Cryptococcus. These methods have been used 1) to clarify the taxonomic relationships among relevant species of the Trichoderma aggregate, 2) to discriminate between aggressive and non-aggressive isolates of the rape seed phytopathogen, Leptosphaeria maculans, and 3) to identify strains of the pathogenic yeasts, Cryptococcus neoformans and Candida albicans. PCR-fingerprinting allowed serotypes of C. neoformans to be distinguished. The application of DNA- and PCR-fingerprinting to fungal DNA should aid in clarification of their taxonomy and improved diagnosis of mycotic disease.

Base Sequence↗

PubMed2000

Study on the production of 6-pentyl-alpha-pyrone using two methods of fermentation.

The lactone 6-pentyl-alpha-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-alpha-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455 mg/l and 167 mg/l after 96 h and 48 h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters.

Biomass↗

PubMed1998

Fungitoxicity of chemical analogs with heartwood toxins.

Trans-stilbene and tropolone as chemical analogs with naturally occurring fungitoxic heartwood compounds were studied with respect to their fungitoxic potency. While stilbene showed no fungitoxic activity towards the fungi Aureobasidium pullulans var. melanogenum, Penicillium glabrum, and Trichoderma harzianum in the concentrations tested, the minimal inhibiting concentration of tropolone was 10(-3) M for Penicillium glabrum and Trichoderma harzianum, and 10(-5) M for Aureobasidium pullulans var. melanogenum. In all cases, the effect of tropolone was a fungistatic one. Using chemical analogs for assessing the chemical basis of the fungitoxicity of tropolone, this substance proved to be the only compound tested which possesses fungitoxic properties.

Fungi↗

PubMed2002

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts.

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.

Amino Acid Sequence↗

PubMed2005

Degradation of nitrocellulose by fungi.

Three lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei and Chaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus and Trichoderma reesei, only 10%-22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.

Biodegradation, Environmental↗

PubMed1991

The 1,4-beta-D-glucan glucanohydrolases from Phanerochaete chrysosporium. Re-assessment of their significance in cellulose degradation mechanisms.

A physico-chemical, functional and structural characterization, including partial sequence data, of three major 1,4-beta-D-glucan glucanohydrolases (EC. 3.2.1.4) isolated from the culture filtrate of the white-rot fungus Phanerochaete chrysosporium, shows that all three enzymes belong to a single family of cellulases. EG44, pI 4.3, (named after its apparent molecular mass in kDa), shows a clear homology with Schizopyllum commune Endoglucanase I (EGI); whereas EG38, pI 4.9, (named in the same manner) is related more closely to Trichoderma reesei (Trichoderma longibrachiatum) Endoglucanase III (EGIII). EG36, pI 5.6-5.7, is probably an EG38 protein lacking its cellulose binding domain. Strong synergistic action is induced by the enzymes acting in concert with cellobiohydrolases (CBHI and CBHII) from the same organism, indicating a highly effective enzymatic system for cellulose degradation. Controlled proteolysis with papain has allowed a so far unique cleavage of endoglucanases EG44 and EG38 into two domains: a core protein, which virtually lacks the capacity to absorb onto microcrystal-line cellulose but retains full catalytic activity against carboxymethyl cellulose and low molecular weight soluble substrates; and a peptide fragment corresponding to the cellulose binding domain. The latter appears to be of paramount significance in the mechanisms involved in the hydrolysis of microcrystalline cellulose.

Amino Acid Sequence↗

PubMed2003

A screening method for endo-beta-1,4-xylanase substrate selectivity.

Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.

Aspergillus↗

PubMed1988

Domain structure of cellobiohydrolase II as studied by small angle X-ray scattering: close resemblance to cellobiohydrolase I.

Evidence for a domain structure of cellobiohydrolase II (CBH II, 58 kDa) from Trichoderma reesei (Teeri et al., 1987; Tomme et al., 1988) is corroborated by results from SAXS experiments. They indicate a 'tadpole' structure for the intact CBH II in solution (Dmax = 21.5 +/- 0.5 nm; Rg = 5.4 +/- 0.1 nm) and a more isotropic, ellipsoid shape for the core protein (Dmax = 6.0 +/- 0.3 nm; Rg = 2.1 +/- 0.1 nm). The latter was obtained by partial proteolysis with papain which cleaves the native CBH II to give two fragments (Tomme et al., 1988): the core (45 kDa) with the active (hydrolytic) domain and a smaller fragment (11 kDa) coinciding with the tail part of the model and containing the binding domain for unsoluble cellulose. This peptide fragment is conserved in most cellulolytic enzymes from Trichoderma reesei (Teeri et al., 1987). It contains a conserved region (block A) and glycosylated parts (blocks B and B' duplicated and located N-terminally in CBH II). In spite of different domain arrangements in CBH I (blocks B-A at C-terminals) SAXS measurements (Abuja et al., 1988) indicate similar tertiary structures for both cellobiohydrolases although discrete differences in the tail parts exist.

Binding Sites↗

PubMed2003

Expression of the red fluorescent protein DsRed-Express in filamentous ascomycete fungi.

The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codon-optimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRed-Express (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRed-Express was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.

Gene Expression Regulation, Fungal↗