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PubMed2006

Simultaneous quantitative LC-ESI-MS/MS analyses of salicylic acid and jasmonic acid in crude extracts of Cucumis sativus under biotic stress.

Salicylic acid (SA) and jasmonic acid (JA) are plant hormones involved in basal resistance against plant pathogens and also in induced resistance. The aim of this study is to develop a fast and sensitive method to determine simultaneously the levels of both these hormones. The present paper proposes a method that includes hormone extraction with MeOH-H(2)O-HOAc (90:9:1, v/v), evaporation of the extracts, and injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system in multiple reaction monitoring (MRM). Endogenous SA and JA levels in noninfested control cucumber cotyledons were 30.96 and 0.73ngg(-1) fresh weight, respectively. In roots, the levels were 8.31 and 15.82ngg(-1) FW, respectively. In plants treated with the biological control agent Trichoderma asperellum strain T-34, the levels of SA and JA did not differ from control plants. Rhizoctonia solani-diseased cucumber plants showed higher levels of SA and JA compared to noninfested controls (up to 2 and 13-fold higher, respectively). Detection limits for SA and JA were 0.45 and 0.47ngg(-1) fresh weight, respectively. The results of our research include the development of a method that is both fast and highly sensitive in the simultaneous quantitation of SA and JA from crude cucumber plant extracts, avoiding any purification and derivatization steps.

Chromatography, Liquid↗

PubMed2004

Evaluation of solid-state bioconversion of domestic wastewater sludge as a promising environmental-friendly disposal technique.

Natural and environmental-friendly disposal of wastewater sludge is a great concern. Recently, biological treatment has played prominent roles in bioremediation of complex hydrocarbon- rich contaminants. Composting is quite an old biological-based process that is being practiced but it could not create a great impact in the minds of concerned researchers. The present study was conducted to evaluate the feasibility of the solid-state bioconversion (SSB) processes in the biodegradation of wastewater sludge by exploiting this promising technique to rejuvenate the conventional process. The Indah Water Konsortium (IWK) domestic wastewater treatment plant (DWTP) sludge was considered for evaluation of SSB by monitoring the microbial growth and its subsequent roles in biodegradation under two conditions: (i) flask (F) and (ii) composting bin (CB) cultures. Sterile and semi-sterile environments were allowed in the F and the CB, respectively, using two mixed fungal cultures, Trichoderma harzianum with Phanerochaete chrysosporium 2094 (T/P) and T. harzianum with Mucor hiemalis (T/M) and two bulking materials, sawdust (SD) and rice straw (RS). The significant growth and multiplication of both the mixed fungal cultures were reflected in soluble protein, glucosamine and color intensity measurement of the water extract. The color intensity and pH of the water extract significantly increased and supported the higher growth of microbes and bioconversion. The most encouraging results of microbial growth and subsequent bioconversion were exhibited in the RS than the SD. A comparatively higher decrease of organic matter (OM) % and C/N ratio were attained in the CB than the F, which implied a higher bioconversion. But the measurement of soluble protein, glucosamine and color intensity exhibited higher values in the F than the CB. The final pH drop was higher in the CB than the F, which implied that a higher nitrification occurred in the CB associated with a higher release of H+ ions. Both the mixed cultures performed almost equal roles in all cases except the changes in moisture content.

Biodegradation, Environmental↗

PubMed2002

Novel tools for the study of class I alpha-mannosidases: a chromogenic substrate and a substrate-analog inhibitor.

The use of chromogenic substrates for evaluation of class I alpha-mannosidase is described. 2('),4(')-Dinitrophenyl-alpha-D-mannopyranoside allows rapid and sensitive assays of enzymatic activities, e.g., of heterologously expressed alpha-1,2-mannosidase from Trichoderma reesei. Interaction constants of several ligands with alpha-mannosidases from class I and II could also be determined. Furthermore, novel types of inhibitors derived from D-lyxose are presented. Methyl-alpha-D-lyxopyranosyl-(1(')-->2)-alpha-D-mannopyranoside is a potent inhibitor of the alpha-1,2-mannosidase from T. reesei (K(i)=600 microM) and since it probably spans subsites -1/+1, this disaccharide could be valuable in crystallographic studies of class I alpha-mannosidases.

Binding, Competitive↗

PubMed1999

The ion-channel activity of longibrachins LGA I and LGB II: effects of pro-2/Ala and gln-18/Glu substitutions on the alamethicin voltage-gated membrane channels.

Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.

Alamethicin↗

PubMed2002

A sugarcane cystatin: recombinant expression, purification, and antifungal activity.

Plants possess several defense mechanisms against pathogenic attack. One of these defenses is the use of protease inhibitor proteins, which interfere in the development and growth of pathogens. Sugarcane productivity can be impacted by the plant's susceptibility to fungal diseases that result in production losses. A relevant line of investigation, therefore, is into the plant's natural defense mechanisms for the control of phytopathogens using cystatins-proteins that specifically inhibit cysteine proteases. In this paper, we discuss the expression, in Escherichia coli, of a sugarcane cystatin, its purification, antifungal activity, and circular dichroism to monitor correct folding. These studies revealed a secondary structure similar to that of the oryzacystatin I of rice. Moreover, the purified protein proved capable of inhibiting the growth of the filamentous fungus Trichoderma reesei, suggesting that it can also be employed to inhibit the growth of pathogenic sugarcane fungi.

Amino Acid Sequence↗

PubMed1989

Structural changes in cellobiohydrolase I upon binding of a macromolecular ligand as evident by SAXS investigations.

Xylan from Rhodymenia palmata binds to the cellobiohydrolase I from Trichoderma reesei (CBH I) or its core protein, inhibiting their activity. Adsorption onto microcrystalline cellulose (Avicel) is reduced approximately 30% for intact CBH I and nearly 50% for the core, whereas the effects with cellobiose are negligible. Structural changes concomitant with this binding are studied in solution by small angle X-ray scattering. In the "tadpole" structure typical for the CBH I [Abuja et al., 1988] the lengthening of the tail part is the most salient observation when xylan is present which accounts for an increase in Dmax (18.0 to 22.0 nm) and radius of gyration (4.74 to 5.18 nm). When xylan binds to the core the radius of gyration remains nearly unchanged. Here a model can be constructed showing a xylan molecule on the surface of the core protein near the tail part.

Adsorption↗

PubMed2002

Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy-D-glucose from beta-chitin.

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine, GlcNAc) from chitin. Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin and beta-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc and N,N'-diacetylchitobiose ([GlcNAc](2)). The product ratio, GlcNAc:[GlcNAc](2), increased while the total yield decreased as the pH was raised from 3. All of the [GlcNAc](2) produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A. cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL.

Acetylglucosamine↗

PubMed2003

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources.

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.

Acetylglucosamine↗

PubMed1997

Substrate specificity of endoglucanases: what determines xyloglucanase activity?

Endoglucanases from Trichoderma viride differ in their activity and mode of action towards xyloglucans. In order to explain the basis for their different behavior, the number of substrate-binding sites of three endoglucanases (endoI, endoIV, and endoV) were determined using bond cleavage frequencies of both normal and reduced cellodextrins and Ko/K(m). EndoIV differed from other endoglucanases described so far, in having at least nine putative binding sites. The specificities of the three endoglucanases towards various xyloglucans derived from apple fruit and potato were determined. Also, the release of oligosaccharides from these substrates in time was monitored. It was concluded that the endoglucanases prefer to bind unbranched glucosyl residues. Because most xyloglucans are composed of XXXG-type of building units, distant subsites are needed to bind xyloglucan. Having at least nine substrate-binding sites, endoIV seems to be well equipped to degrade xyloglucans which was confirmed by its high xyloglucanase activity.

Carbohydrate Sequence↗

PubMed1979

Analysis of cellulase proteins by high-performance liquid chromatography.

A new procedure using high-performance liquid chromatography for the rapid separation of cellulase proteins is described. The cellulase components of Trichoderma reesei are fractionated on a DEAE anion-exchange column using a phosphate buffer at pH 6.2. Activities of the individual components obtained from T. reesei QM6a, a wild strain, and several mutant strains have been determined. Each system examined contained beta-glucosidase, at least two exo-beta-1,4 glucanases and five endo-beta-1,4 glucanases with the endo-beta-1,4 glucanases accounting for 20--36% and the exo-beta-1,4 glucanases for 64--80% of the soluble protein.

Cellulase↗

PubMed1999

Discrimination between enantioselective and non-selective binding sites on cellobiohydrolase-based stationary phases by site specific competing ligands.

A systematic study was performed to investigate the influence of cellobiose or lactose on the enantioselective retention behaviour of some beta-blockers in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderma reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobilized on silica as stationary phases. The results revealed that the retention could be described by the function [equation; see text] where the observed capacity factor corresponds to the sum of an enantioselective mode being influenced by a site specific competing ligand (competitor) and a non-selective mode unaffected by the competitor. A non-constrained non-linear least-square regression gave in all cases virtually identical nondisplacable capacity factors (k'ns) for both enantiomers of the same drug. The experimental capacity factors (k'(x,C)) of the enantiomers all show a close fit to the adapted function. The Kd values calculated for the competitor were also virtually identical for each pair of enantiomers and were in accordance with Ki data determined for the competitors in classical enzyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar results were obtained with the resolution of rac-alprenolol and rac-metoprolol on CBH I phase.

Binding Sites↗

PubMed2003

Ginsenosides stimulate the growth of soilborne pathogens of American ginseng.

Ginseng saponins (ginsenosides) were isolated from soil associated with the roots of commercially grown American ginseng (Panax quinquefolius L.), identified via LC-MS and quantified via analytical HPLC. The ginsenosides, including F(11), Rb(1), Rb(2), Rc, Rd, Re and Rg(1), represented between 0.02 and 0.098% (average 0.06%) of the mass of the soil collected from roots annually between 1999 and 2002. The same ginsenosides were also isolated from run-off of undisturbed plants grown in pots in a greenhouse using a root exudate trapping system. To investigate (1) whether these saponins could influence the growth of pythiaceous fungi pathogenic to ginseng, and (2) whether soil levels of ginsenosides were sufficient to account for any effects, bioassays were completed using a crude saponin extract and an ecologically relevant concentration of purified ginsenosides. Thus, when cultured on media containing crude saponins, the colony weight of both Phytophthora cactorum and Pythium irregulare was significantly greater than that of control, indicating a strong growth stimulation by ginsenosides. The growth of Pythium irregulare was also significantly stimulated after addition of an ecologically relevant, low concentration (i.e. 0.06%) of purified ginsenosides to culture medium. By contrast, growth of the saprotrophic fungus Trichoderma hamatum was slightly (but not significantly) inhibited under the same conditions. These results imply that ginsenosides can act as allelopathic stimulators of the growth of pythiaceous fungi in the rhizosphere, and this may contribute to the disease(s) of this crop.

Dose-Response Relationship, Drug↗

PubMed1998

The cystine knot structure of ion channel toxins and related polypeptides.

An increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded beta-sheet in toxic and inhibitory polypeptides. Protein Science 3, 1833-1839). These globular, disulfide-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel beta-sheet. Examples of ion-channel toxins known to adopt this structure are the omega-conotoxins and omega-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as kappa-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exemplified by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, delta-conotoxin PVIA and the muO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disulfide bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaffold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets.

Agatoxins↗

PubMed1997

Screening of fungal beta-xylanases for production of acidic xylooligosaccharides using in situ reduced 4-O-methylglucuronoxylan as substrate.

Fungal beta xylanases were screened for production of acidic xylooligomers from 4-O-methylglucuronoxylan. In situ reduced hardwood xylan was used as substrate because the products of neutral- and acidic-branched xylooligomers help define substrate specificity of the enzymes. Borohydride reduction in situ transformed 30% of 4-O-methyl-alpha-D-glucopyranosyluronic acid residues into 4-O-methyl-alpha-D-glucopyranosyl residues and reduced C-1 end groups in the xylan backbones. A total of ten beta-xylanase fractions from four fungi were partially purified by chromatography by anion exchange and molecular sieving, and graded qualitatively for enzymatic hydrolysis of the substrate. The yield of acidic xylooligomers was highly affected by whether alpha-glucuronidases were present in the beta-xylanase fractions. Some fractions gave free 4-O-methyl-alpha-D-glucopyranosyluronic acid, but none of the enzyme fractions could release free 4-O-methyl-alpha-D-glucose. Among the beta-xylanase fractions studied, xylanase II of Trichoderma viride was the best producer of aldotetraouronic acid [2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylotriose]. The results obtained suggested that there was a difference in the steric hindrance of the branch point on fungal beta-xylanases between 4-O-methyl-alpha-D-glucopyranosyl and 4-O-alpha-D-glucopyranosyluronic acid residues.

Basidiomycota↗

PubMed2001

A convenient fluorometric method for the detection of extracellular N-acetylglucosaminidase production by filamentous fungi.

A rapid and convenient method for the detection of chitinases accumulating in filamentous fungal cultures was developed. The assay is performed on cultures growing in microtiter plates, with a fluorogenic substrate: 4-methylumbelliferyl-N-acetyl-D-glucosaminide (4-MeUNAG). The fluorescence of the product, 4-methylumbelliferone, was detected. This method was successfully used to follow induction and repression of extracellular exochitinase activity in the biocontrol fungus Trichoderma harzianum.

Acetylglucosaminidase↗

PubMed2003

Metabolic activity in filamentous fungi can be analysed by flow cytometry.

The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and enumerate bacterial and yeast cells is well established. We have shown that through the judicial choice of stains, the nondestructive screening and sorting of fungal material is possible. The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma, have been followed using forward- and side-angle scatter on a Becton Dickinson FACSCalibur flow cytometer. By staining isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured. We have been able to demonstrate that there is a 12-13 h window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry.

Antarctic Regions↗

PubMed2001

Study of protein adsorption on indigo particles confirms the existence of enzyme--indigo interaction sites in cellulase molecules.

Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57-111 and 0-62 microg x mg(-1), respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.

Adsorption↗

PubMed2001

Application of microassays for investigation of cellulase abrasive activity and backstaining.

Model microassays were used for testing the denim-washing performance and indigo backstaining for Trichoderma reesei and Chrysosporium lucknowense commercial cellulase preparations on a 'test-tube scale'. C. lucknowense preparation demonstrated a higher potential in the denim biostoning process. The performance of four purified cellulases (two endoglucanases and two cellobiohydrolases) from C. lucknowense on cotton textiles was assayed, and the key enzyme (endoglucanase with a molecular weight of 25 kDa) responsible for high abrasion effects on denim fabrics was found.

Cellulase↗