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Crystallization and preliminary X-ray diffraction studies of the catalytic core of acetyl xylan esterase from Trichoderma reesei.

Acetyl xylan esterase is involved in the biodegradation of hemicellulose. It cleaves O-acetyl groups from xylan, which is the most abundant hemicellulose in nature. The catalytic core of acetyl xylan esterase from T. reesei has been crystallized and X-ray diffraction data at 2.3 A collected. The crystal belongs to the triclinic space group P1 with unit-cell parameters a = 50.3, b = 62. 1, c = 40.0 A, alpha = 110.1, beta = 113.6 and gamma = 97.9 degrees. The asymmetric unit contains two molecules.

Acetylesterase↗

Properties of uracil transport by vegetative mycelium of Trichoderma viride.

The transport of radioactively labelled uracil into submerged mycelium of T. viride was measured by means of a membrane filtration technique. It was found to be time-dependent (up to 90 min) and concentration-dependent (up to 8 mmol l-1). Its concentration dependence was biphasic and consisted from the saturatable part (at the uracil concentration below 0.2 mmol l-1) with KM = 0.08 +/- 0.02 mmol l-1 and Vmax = 1.74 +/- 0.3 nmol (mg dry wt.)-1 h-1, and from the region at higher uracil concentration which showed only a weak saturatability with the substrate. The transport measured in the saturatable part of the curve was also pH- and temperature-dependent. The optimal pH was between 5.4 and 6.4 and the optimal temperature was at 37 degrees C. The activation energy of 54 kJ mol-1 and the temperature quotient of Q10 = 2.1 could be calculated from the temperature dependence. The entry of uracil was in part inhibited by nucleobases and their analogues, nucleosides, nucleotides and amino acids. The inhibitors had similar inhibitory efficiency about 50% at 0.2 mmol l-1. 3,3',4',5-tetrachlorosalicylanilide (TCS), the uncoupling agent, significantly inhibited the uracil transport, but its inhibitory efficiency decreased upon increasing the uracil concentration. Ionophore antibiotics valinomycin and monensin also inhibited the uracil transport. Inhibitors of RNA-polymerase, rifamycin and rifampicin were without effect. The results suggest that at low uracil concentrations (below 0.2 mmol l-1), its transport is mediated by a carrier and is driven by the electrochemical potential of protons. At higher uracil concentrations, the transport may be driven by the concentration difference of uracil with the contribution of the protonmotive force. It is feasible that inhibitors of uracil transport tested exert their inhibition by the dissipation of the driving force rather than by the direct competition with the substrate-binding site.

Biological Transport↗

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.

Animals↗

Biosynthesis of bisorbicillinoid in Trichoderma sp. USF-2690; evidence for the biosynthetic pathway, via sorbicillinol, of sorbicillin, bisorbicillinol, bisorbibutenolide, and bisorbicillinolide.

An incorporation study of [1-(13)C] and [1,2-(13)C2] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using 13C-labeled 1 prepared from [1-(13)C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated 13C-labeled 1 into their carbon skeletons. 13C-labeled bisorbicillinols 2 derived from [1-(13)C]- and [2-(13)C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.

4-Butyrolactone↗

Enzyme production of Trichoderma reesei Rut C-30 on various lignocellulosic substrates.

Economical production of cellulase enzyme is key for feasible bio-ethanol production from lignocellulosics using an enzyme-based process. On-site cellulase production can be more feasible with the process of separate hydrolysis and fermentation (SHF) than with simultaneous saccharification and fermentation, since the cost of enzyme is more important and a variety of substrates are available for the SHF process. Cellulase production using various biomass substrates available for SHF, including paper sludge, pretreated wood (steam exploded), and their hydrolysis residues, was investigated in shake flasks and a fermenter for their productivities and titers. Among the newspaper sludge, office paper sludge, and steam-exploded woods treated in various ways, the steam-exploded wood showed the best properties for substrate in cellulase production. The best titer of 4.29 IU/mL was obtained using exploded wood of 2% (w/v) slurry in the shake flask, and the titer with the same substrate was duplicated to about 4.30 IU/mL in a 3.7-L fermenter. Also, the yield of enzyme reached 215 IU/g of substrate or 363 IU/g of cellulose. Despite various pretreatment attempts, newspaper and office paper substrate was inferior to the exploded-wood substrate for cellulase production. However, hydrolysis residues of papers showed quite promising results. The hydrolysis residue of office paper produced 2.48 IU/mL of cellulase in 7 d. Hence, the utilization of hydrolysis residues for cellulase production will be further investigated in the future.

Biomass↗

The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma.

M14-2 is a cellulase hyperproducer derived from Trichderma reesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.

Cellulase↗

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Biotechnology↗

Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters?

Trichoderma/Hypocrea is a genus of soil-borne or wood-decaying fungi containing members important to mankind as producers of industrial enzymes and biocontrol agents against plant pathogens, but also as opportunistic pathogens of immunocompromised humans. Species identification, while essential in view of the controversial properties of taxa of this genus, has been problematic by traditional methods. Here we will present a critical survey of the various identification methods in use. In addition, we will present an update on the taxonomy and phylogeny of the 88 taxa (which occur as 14 holomorphs, 49 teleomorphs and 25 anamorphs in nature) of Trichoderma/Hypocrea that have been confirmed by a combination of morphological, physiological and genetic approaches.

Chromosome Mapping↗

Toxic mold and mycotoxins in neurotoxicity cases: Stachybotrys, Fusarium, Trichoderma, Aspergillus, Penicillium, Cladosporium, Alternaria, Trichothecenes.

Presented is the argument that psychologists and neuropsychologists have no scientific basis for rendering opinions about causation given the current state of the literature. The critical question is whether in a residence or office inhalation of mold spores or mold metabolites, including mycotoxins, causes neuropsychological impairment or mental and emotional disorders. There has not been sufficient research to support such conclusions. Nonetheless, in the context of litigation, speculative opinions are rendered in lieu of scientifically well-founded conclusions. Resources for recognizing and coping with pseudoscientific arguments are suggested.

Alternaria↗