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PubMed1991

Determination of the cellulolytic activity of Micromycetes by an express method.

An express method of testing the cellulolytic activity of Trichoderma viride in media with cellulose-containing substrates by the rate of glucose production has been elaborated. The optimum temperature selections and duration of sample incubation (1 h, 50 degrees C) have been described, and the temperature coefficient for the scale-down of the enzyme complex from 50 to 30 degrees C (0.77) has been determined.

Cellulase↗

PubMed1976

Rhizosphere mycoflora of wheat after foliar application of chlorocholine chloride, urea and 4-chloro-2-methylphenoxyacetic acid.

A single-step spraying of wheat during shooting under field conditions with solutions of CCC (chlorocholine chloride), CCC and urea, CCC and Aminex (ammonium salt of 4-chloro-2-methylphenoxyacetic acid), or CCC, urea and Aminex caused changes both in numbers and composition of the rhizosphere mycoflora. The numbers both in the rhizosphere of differently treated plants and in the free soil decreased during vegetation. A more pronounced effect in the number of fungi was demonstrated in plants treated only with CCC. The difference was more considerable during first 10 days after the spray. As far as the relative occurrence of individual genera in the rhizosphere soil is concerned, fungi of the genera Penicillium Link ex Fr., Fusarium Link ex Fr., Verticillium Nees and Trichoderma Pers were most influenced after the treatment with the used agents.

Choline↗

PubMed1978

Effect of volatile substances from Origanum majorana and Ocimum basilicum on spore respiration and germination of some soil fungi.

Oxygen uptake by the spores of Fusarium moniliforme, F. oxysporum, F. semitectum, F. solani, Mucor racemosus and Trichoderma viride was increased in the presence of volatile substances extracted from Origanum majorana and Ocimum basilicum. This increase was greater in the presence of volatile substances from O. basilicum than O. majorana, except in the case of F. semitectum where the reverse was true. A drop in the RQ of all the germinating spores was observed in the presence of these substances. Volatile substances from O. majorana reduced the spore germination of M. racemosus whereas the spores of T. viride were stimulated to germinate. Volatile substances from O. basilicum stimulated the spore germination of M. racemosus whereas T. viride spores were not affected.

Fusarium↗

PubMed1998

Biosorption of nickel using filamentous fungi.

Nickel (Ni) uptake capability from aqueous solutions was studied in a filamentous fungi strains group of Rhizopus sp., Penicillium sp. Aspergillus sp., Trichoderma sp., Byschoclamyss sp., and Mucor sp. The metal uptake of a Rhizopus sp. strain, which has the highest uptake capacity, was corroborated by electron microscopy; no Ni deposits were observed on the cell wall, but rather a homogeneous accumulation was seen on the cell surface. The influence on the capacity of metal uptake by environmental parameters such as pH, temperature, time, and the interference of other ions in the solution, was also studied. Nickel accumulation by the selected strains is fast, occurring in less than 30 min, and does not require a microorganism's active metabolism to take place. The sorption isotherms were established for the selected fungi, in order to determine the maximum metal uptake capacity. The sorption isotherms were fixed to the mathematical models of Freundlich and Langmuir, obtaining better performance on the Langmuir model.

Aspergillus↗

PubMed1991

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis.

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase from Trichoderma viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.

Adsorption↗

PubMed1980

Volatile and gaseous metabolites released by germinating seeds of lentil and maize cultivars with different susceptibilities to fusariosis and smut.

The effect of volatile and gaseous metabolites released by germinating seeds of lentil cultivars more and less susceptible to fusariosis on the germination of spores of Mucor racemosus, Trichoderma viride, Verticillium dahliae and Botrytis cinerea was found to depend rather on the fungal genus than on the lentil cultivar. However, spores of Fusarium oxysporum reacted more sensitively during germination to the presence of exudates of both cultivars, when the more susceptible lentil displayed a stimulation, the less susceptible one an inhibition of spore germination. The greatest difference in the effect of exudates was observed in the more and less susceptible maize cultivars with respect to the germination of chlamydospores of Ustilago maydis, especially during the first hours of seed germination. Analysis of the exudates of germinating seeds showed the release of a greater amount of ethanol and methanol with acetaldehyde by the more susceptible cultivars of lentil and particularly maize.

Ethanol↗

PubMed1991

The use of some fluorescent stains for studying morphogenesis of micromycetes.

The use of some classical fluorochromes and optical brighteners in the fluorescence microscopy of micromycetes was investigated. Of the 16 compounds tested on slide cultures of Trichoderma viride 3 were too toxic, whereas the other stained primarily hyphae with various intensity. Reproductive structures did not stain or stained only weakly. With respect to vital staining the optical brightener Blankophor RKH exhibited most favorable properties. It did not inhibit either the growth or sporulation and stained intensively hyphae, septa and growth apices in particular. It also induced intensive fluorescence of a growing yeast culture.

Fluorescent Dyes↗

PubMed2005

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences.

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.

Aspergillus niger↗

PubMed2002

Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol.

The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.

Cellulose↗

PubMed2002

Enzymatic hydrolysis and physical characterization of commercial celluloses and cellulose-based ion-exchange powdered mixed resins.

Commercial celluloses (BH20, Epicote, FC+) and their cellulose-containing powdered mixed resins (PMR) were evaluated using enzymatic and physical methods. Samples were hydrolyzed with purified Trichoderma viride cellulase extract and measured for released reducing sugar using the dinitrosalicylic acid method. Physical characterization was performed with gross specific surface areas (GSSA) and relative crystalline indices (RCI). In addition, FC+ was exposed to physical and chemical processing commonly encountered in spent PMR processing to determine potential effects on reducing sugar release in high intensity containers. Reducing sugar released from the celluloses by T. viride cellulase ranged from 135.37 to 244.48 mg day(-1); the celluloses were highly crystalline, ranging from 82.47 to 84.57%; and the GSSA medians for the celluloses ranged from 1,298.60 cm(2) g(-1) to 2,493.20 cm(2) g(-1). Most processing treatments on the FC+ reduced the amount of reducing sugar released and increased RCI. Cellulose hydrolysis rates did not show a strong correlation with the physical characterization. These results suggest that (1) celluloses and PMR can serve as abundant sources of bioavailable carbon in water treatment systems, and (2) the use of correlative physical characteristics to evaluate a cellulose-based commercial product may not accurately predict microbial activity; a complementary microbial test such as cellulose hydrolysis with cellulase may prove useful.

Cellobiose↗

PubMed2004

Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain.

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.

Amino Acid Sequence↗

PubMed2005

Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083.

Arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis releases only C3-linked arabinose residues from double-substituted xylose residues. A genomic library of B. adolescentis DSM20083 was screened for the presence of the axhD3 gene. Two plasmids were identified containing part of the axhD3 gene. The nucleotide sequences were combined and three open reading frames (ORFs) were found. The first ORF showed high homology with xylanases belonging to family 8 of the glycoside hydrolases and this gene was designated xylA. The second ORF was the axhD3 gene belonging to glycoside hydrolase family 43. The third (partial) ORF coded for a putative carboxylesterase. The axhD3 gene was cloned and expressed in Escherichia coli. Several substrates were employed in the biochemical characterization of recombinant AXHd3. The enzyme showed the highest activity toward wheat arabinoxylan oligosaccharides. In addition, beta-xylanase from Trichoderma sp. was able to degrade soluble wheat arabinoxylan polymer to a higher extent, after pretreatment with recombinant AXHd3. Arabinoxylan oligosaccharides incubated with a combination of recombinant AXHd3 and an alpha-L-arabinofuranosidase from Aspergillus niger did not result in a higher maximal release of arabinose than incubation with these enzymes separately.

Arabinose↗

PubMed2005

Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan.

The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1-->4)-beta-D-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5' form of diferulic acid from arabinoxylan (AX) derived from brewers' spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.

Ascomycota↗

PubMed2006

Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases.

In the present study, we modified xylose uptake properties of a recombinant xylose-utilizing yeast Saccharomyces cerevisiae by expression of heterologous and homologous permease-encoding genes. In a mutant yeast strain with the main seven hexose transporter genes deleted, and engineered for xylose utilization, we screened an expression cDNA library of the filamentous fungus Trichoderma reesei (Hypocrea jecorina) for enhanced growth on xylose plates. One cDNA clone with significant homology to fungal sugar transporters was obtained, but when the clone was retransformed into the host, it did not support significant growth on xylose. However, during a long liquid culture of the strain carrying the cDNA clone, adaptive mutations apparently occurred in the host, which led to growth on xylose but not on glucose. The new transporter homologue, Trxlt1 thus appears to code for a protein specific for xylose uptake. In addition, xylose-transporting properties of some homologous hexose transporters were studied. All of them, i.e., Hxt1, Hxt2, Hxt4, and Hxt7 were capable of xylose uptake. Their affinities for xylose varied, K (m) values between 130 and 900 mM were observed. The single-Hxt strains showed a biphasic growth mode on xylose, alike the Trxlt1 harboring strain. The initial, slow growth was followed by a long lag and finally by exponential growth.

Evolution, Molecular↗

PubMed1997

The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds.

The ability of a wide variety of soil-borne fungal strains to degrade four structurally different compounds containing P-C bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)methylphosphonic acid effectively.

Alanine↗

PubMed1999

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti.

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus.

Amino Acid Sequence↗

PubMed2003

The application of PCR for the isolation of a lipase gene from the genomic DNA of an Antarctic microfungus.

We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes. We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium. The recombinant lipase had a temperature optimum of 25 degrees C at pH 7.9 and retained greater than 50% of the maximum activity from 10 degrees C to 35 degrees C and over a pH range from 4.0 to 8.5.

Amino Acid Sequence↗

PubMed2000

Molecular characterization of a PDI-related gene prpA in Aspergillus niger var. awamori.

A gene (prpA) homologous to the protein disulfide isomerase gene was isolated from Aspergillus niger by Southern hybridization using the pdi1 gene isolated from Trichoderma reesei as a DNA probe. The corresponding cDNA of the prpA gene has also been isolated from an A. niger var. awamori cDNA library. The prpA gene does not belong to any currently recognized family of protein disulfide isomerases since it contains only a single conserved thioredoxin domain at the N-terminus of the protein. The C-terminal two-thirds of the protein has no homology to any known proteins in the database. The PRPA protein contains an ER retention signal (HDEL) at its C-terminal end suggesting that it is located in the ER. Southern hybridization at high stringency showed that it was present as a single copy in the genome. Northern hybridization indicated that the transcript level of the prpA gene was higher if the cells were secreting a heterologous protein, bovine prochymosin. However, over-expression of the prpA gene from a multicopy integrated vector had little effect on chymosin secretion. A strain containing a deletion of the prpA gene was viable. However, deletion of the prpA gene appeared to cause a reduction of bovine chymosin production.

Amino Acid Sequence↗