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PubMed2006

Efficient isolation of anthraquinone-derivatives from Trichoderma harzianum ETS 323.

Anthraquinone-derivatives, chrysophanol and pachybasin, were purified by a silica column chromatography with two different solvent systems from Trichoderma harzianum ETS 323. The fungus was incubated in sugarcane bagasse solid medium at room temperature without rotation. Structure of chrysophanol was solved by X-ray diffraction and pachybasin by NMR spectra. About 233+/-13 mg of pure chrysophanol and 773+/-40 mg of pure pachybasin were recovered per kg of solid cultural medium, with yields 1.7+/-0.2% and 5.6+/-0.5%, respectively.

Anthraquinones↗

PubMed2004

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin.

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.

Adsorption↗

PubMed2004

A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II.

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.

Amino Acid Substitution↗

PubMed2004

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton.

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).

Cellulose 1,4-beta-Cellobiosidase↗

PubMed2005

Production of glucuronan oligosaccharides using a new glucuronan lyase activity from a Trichoderma sp. strain.

Sinorhizobium meliloti M5N1CS synthesizes a homopolymer of glucuronic acids beta-(1,4) linked and variably C2 and/or C3O-acetylated. To obtain beta-Delta-(4,5)-unsaturated oligoglucuronans, various acetylated forms of this bacterial polymer were cleaved by a Trichoderma sp. GL2 glucuronan lyase. Oligomers with polymerization degrees up to 8 were then produced, purified by liquid chromatography (size exclusion and anions exchange) and characterized using 1H NMR and ESI-Q/TOF-MS. Finally, the production (in gram quantity) of pure unsaturated oligoglucuronans non-acetylated (di- and trisaccharide) was investigated thanks to the complete depolymerization of deacetylated glucuronan.

Acetylation↗

PubMed2005

Increased alkali stability in Trichoderma reesei endo-1, 4-beta-xylanase II by site directed mutagenesis.

A number of engineered Trichoderma reesei endo-beta-1,4-xylanase (Xyn II) mutants were created and activity tests were performed for increased stability. The stability of the earlier characterized mutant Y5 (T2C, T28C, K58R, +191D) was further increased by the mutations creating the constructs P9 (N97R+F93W+H144K), P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C). The resistance towards thermal inactivation at alkaline pH was increased in all of the mutants. Residual activity T(50%) was increased 4-5 degrees C for P9 at pH 9. The performance of the P9 mutant in sulphate pulp bleaching was also tested and was shown to increase brightness markedly compared to the reference. The bleaching results showed the industrial potential of the obtained mutant.

Alkalies↗

PubMed2006

Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum.

An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.

Enzyme Stability↗

PubMed2006

Exploitation of Trichoderma harzianum mycelial waste for the removal of rhodamine 6G from aqueous solution.

The harvested mycelial waste of Trichoderma harzianum was used as an adsorbent for the removal of rhodamine 6G and was studied in batch mode. The effects of agitation time and initial dye concentration, adsorbent dosage and pH were examined. The study revealed that the amount of dye adsorbed (mgg(-1)) increased with increase in agitation time and reached equilibrium after 120 min, for dye concentrations of 10-50 mg L(-1). The adsorbent dosage of 1.0 g/50 mL and pH of 8.0 were found to be optimum for maximum dye removal. The batch mode adsorption data followed both the Langmuir and Freundlich isotherms. The pseudo first- and second-order rate kinetics were applied to the adsorbent system. The adsorption kinetics of rhodamine 6G showed that the pseudo-second-order kinetic model provided the best correlation of the equilibrium data. The study implies that it is possible to develop a dye removal system by using T. harzianum biomass, which occurs as sludge in waste stream of fermentation industries.

Adsorption↗

PubMed2004

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Binding Sites↗

PubMed2005

Statistical analysis of structural and kinetic characteristics of fungal colony growth with Trichoderma viride PERS.: S.F. Gray.

The mycelial colonies of Trichoderma viride were grown between two thin cellophane films for exact measurements. The results obtained testify to the fact that in a mature colony the average length of intercalary cells, the average number of intercalary cells in an internode and the average internode length are stable. At this stage of morphogenesis the mean internode length is shown to be equal to the product of the average intercalary cell length and the average number of intercalary cells in an internode. The coefficients of variation of the internode length and the number of intercalary cells in an internode are found to be equal. The length of an intercalary cell and duration of the doubling cycle of an apical cell of T. viride obey the law of Gamma distribution. According to our observations, the Gamma distribution is typical for the length and duration of the doubling cycle of any bacterial cells and cells of any multicellular organisms.

Culture Media↗

PubMed2005

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain.

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.

Basidiomycota↗

PubMed2004

Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii.

The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 512 IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.

Amino Acid Sequence↗

PubMed2006

Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli.

Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.

Chitinases↗

PubMed2004

Regulation of N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum: evidence that cAMP controls its expression.

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-beta-D-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription.

Acetylglucosaminidase↗

PubMed2005

Wastewater sludge as a potential raw material for antagonistic fungus (Trichoderma sp.): role of pre-treatment and solids concentration.

Feasibility of production of antagonistic Trichoderma sp. conidial spores using wastewater sludge as a raw material employing different suspended solids concentration (10-50 g/l) was investigated in shake flasks. Maximum conidial spore count obtained for raw sludge was 1.98 x 10(4) CFU/ml, which was enhanced by sludge pre-treatments (alkaline and thermal alkaline). Conidial spore count ranging from 1.3 x 10(6) to 2.8 x 10(7) CFU/ml was observed for alkaline and thermal alkaline treated sludges. Optimal suspended solids concentration was 30 g/l (10(7) CFU/ml) whereas, lower (<20 g/l) and higher (>30 g/l) solids concentration were less efficient. Thermal alkaline pre-treated sludge showed diauxic growth due to multiplicity of sludge biodegradability. A simple, modified CFU filtration technique was also developed for fungal spore assessment in sludge. Bioassay of fermented sludge against spruce budworm larvae showed entomotoxicity (15036 SBU/microl), on par with Bacillus thuringiensis biopesticides. This study successfully demonstrated potential of wastewater sludge as a raw material for production of value added product, aiding in sludge management and proliferation of eco-friendly and economical biocontrol agents.

Animals↗

PubMed2004

Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei: justification of mycotoxin testing in food grade enzyme preparations derived from the three fungi.

Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei are three important production organisms used in industrial fermentations. Several of the fungal secondary metabolites produced by selected strains of these three fungi are capable of eliciting toxicity in animals. Among those toxic substances are the well-known mycotoxins 3-nitropropionic acid and ochratoxin A. However, many others, such as kojic acid, may not be true mycotoxins. The production, extraction, chemical structure, and the toxicity (expressed as LD(50)) of these substances are reviewed. Production of toxic secondary metabolites in A. niger, A. oryzae, and T. reesei is strain-specific and environment-dependent. Considering all of the safety measures taken in the industrial production process, these three fungal species are safe to use. The recently revised JECFA specification for mycotoxins in food enzyme preparations is also discussed. The extent of mycotoxin tests in food enzyme preparations should be judged on a case-by-case basis, through a careful evaluation based on knowledge of taxonomy, biochemistry, and genetics. In many cases, the testing scope at the level of genus should be sufficient. In other cases, the scope can even be further narrowed based on scientific knowledge and assessment.

Animals↗

PubMed2002

Aggregation and self-assembly of hydrophobins from Trichoderma reesei: low-resolution structural models.

Hydrophobins are secreted fungal proteins, which have diverse roles in fungal growth and development. They lower the surface tension of water, work as adhesive agents and coatings, and function through self-assembly. One of the characteristic properties of hydrophobins is their tendency to form fibrillar or rod-like aggregates at interfaces. Their structure is still poorly known. In a step to elucidate the structure/function relation of hydrophobin self-assembly, we present the low-resolution structure of self-assembled fibrils of the class II hydrophobin HFBII from Trichoderma reesei based on small and wide-angle x-ray scattering. We first studied the solution state (10 mg/mL) of both HFBI and HFBII and showed that they formed assemblages in aqueous solution, which have a radius of gyration of ~24 A and maximum dimension of ~65 A, corresponding to the size of a tetramer. This result was supported by size-exclusion chromatography. Undried samples of HFBII fibrils had a monoclinic crystalline structure, which changed to hexagonal when the material was dried. A low-resolution structure for the HFBII fibrils is suggested. There are data in the literature based on staining properties suggesting that hydrophobins of class I form assemblies with an amyloid structure. Comparison of the HFBII data (x-ray results, staining with thioflavin T) to published data showed that the HFBII assemblages are not amyloid.

Benzothiazoles↗

PubMed2003

New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins.

Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases.

Acetylation↗