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PubMed2001

Cellulase finishing of woven, cotton fabrics in jet and winch machines.

Some authors have reported that as the applied agitation rate increases, the apparent activity of the endoglucanases from Trichoderma reesei towards cotton cellulose increases more markedly than does the apparent activity of the cellobiohydrolases. This suggests that the quality of cellulase finishing effects on cellulosic textiles may be machine-type dependent. The present work using total crude, endoglucanase-rich and cellobiohydrolase-rich cellulases from T. reesei confirmed that the final properties of woven, cotton fabrics treated under realistic processing conditions in a jet machine, were measurably and perceivably different from those of the same fabrics, treated using the same processing conditions of temperature, time, pH, enzyme concentration and fabric to liquor ratio, but in a winch machine. The results are interpreted in terms of the effects of agitation rate on the adsorption-desorption behaviour of the T. reesei endoglucanases and cellobiohydrolases.

Cellulase↗

PubMed2002

Studies on the properties of Celluclast/Eudragit L-100 conjugate.

A cellulase from Trichoderma reesei was immobilized on Eudragit L-100, a reversibly soluble polymer depending on the pH of the medium. The solubility of the modified cellulase was studied at different pH values. By changing the pH, the adsorption equilibrium of the derivatized proteins is switched towards the liquid phase, thus making recycling possible. This method allows for improved stability, without major loss of specific activity. The adsorption of cellulase on Eudragit lowers the enthalpy of denaturation, but affects only slightly the denaturation temperature. The use of carbodiimide was ineffective on linking the enzymes covalently to the polymer, since the immobilization process was found to be only mediated by non-covalent forces.

Adsorption↗

PubMed2003

Safety evaluation of a glucanase preparation intended for use in food including a subchronic study in rats and mutagenicity studies.

An enzyme preparation containing glucanase produced by the fungus Trichoderma harzianum is intended to be used to improve the clarification and filtration of wines. The safety of a glucanase preparation was assessed in a series of toxicological tests to document its safety in use. Oral administration to rats of up to 10 mL/kg bw/day (equivalent to a total organic solids dosage of 1258 mg/kg bw/day or a glucanase dosage of 1882 BGXU/kg bw/day) for 13 weeks did not cause any adverse effect. The test substance was not found to be mutagenic in the bacterial reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes. The safety margin for exposure is estimated to be in the range of 2100-72,000 depending on the estimate for daily wine consumption. The results of these studies demonstrate that an enzyme preparation containing glucanase may be considered safe when employed in wine processing.

Administration, Oral↗

PubMed2000

The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy.

Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented.

Cellulase↗

PubMed2002

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.

Cellulase↗

PubMed2003

Efficient synthesis of glyceroyl beta-lactoside and its derivatives through a condensation reaction by cellulase.

Condensation reaction between lactose and glycerol was effectively catalyzed by utilizing a commercially available cellulase preparation from Trichoderma reesei. The enzyme induced the formation of 1-O-beta-lactosyl-(R,S)-glycerol (1) and 2-O-beta-lactosyl glycerol (2) in a molar ratio of 7:3 and in a 20% yield based on lactose added. The enzyme also induced the condensation of lactose with 1,3-propanediol to produce O-beta-lactosyl propanediol (3) in a yield of 15%. When various alkanols (N: 2-8) and allyl alcohol were used in the condensation reaction, the corresponding alkyl and allyl beta-lactoside were obtained in the yields of 0.9-3.8% of the desired compounds.

Cellulase↗

PubMed2000

Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction.

Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E. coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol-phosphate systems. The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems. A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared. The methods differ in the height of the optimum found, the number of experiments and the time required. The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems. The simplex procedure has to be further improved.

Algorithms↗

PubMed2000

Effect of biological suspensions on the position of the binodal curve in aqueous two-phase systems.

This study is concerned with the influence of biological suspension on the position of the binodal curve in aqueous two-phase systems (ATPSs). Three different biological suspensions (i.e., disrupted yeast, E. coli homogenate and fermentation broth from Trichoderma harzianum) were selected and their impact upon ATPS performance was evaluated on the basis of changing volume ratio (Vr) and the position of the binodal curve of biological ATPSs (added with biomass). Biological ATPSs with initial Vr greater than 1 and long (>40%, w/w) tie-line length (TLL), exhibited significant changes in Vr when compared with that from non-biological systems. Such behaviour was associated with the top phase biomass accumulation. It was shown that the addition of the biological suspensions used in this study to ATPSs caused the binodal curve to displace towards the origin, which was associated with the critical contribution of the bio-polymer (present in the systems) to the phase formation. The practical implementation of ATPSs for the purification of biological materials exploiting the information reported in this study is discussed.

Escherichia coli↗

PubMed1998

Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii.

The chitinolytic activity of Verticillium cfr. lecanii A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 degrees C in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15 degrees C, the levels of enzyme activity of the three strains were similar to those obtained at 25 degrees C. At 5 degrees C, in contrast, the activity of V. lecanii was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h). The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60 degrees C): at 5 degrees C, its relative activity was still 50% of that recorded at 40 degrees C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by mycelial damage and cell lysis.

Antarctic Regions↗

PubMed2001

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast.

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.

Carbohydrate Metabolism↗

PubMed2002

SCP and crude pectinase production by slurry-state fermentation of lemon pulps.

Single cell protein (SCP) and crude pectinolytic enzymes production from citrus pulps is reported. SCP and enzymes were produced by slurry-state flask cultivation of Aspergillus niger and Trichoderma viride on pulps from lemon juice clarification. Production as well as crude pectinase activity was not affected by the high dry matter content of the pulps. Both the protein content in the residue and the enzyme activity in the supernatant were higher in T. viride than in A. niger culture. The crude pectinase of T. viride, whose specific activity was similar to that found for a commercial concentrated preparation, could be utilized in the same citrus processing factory as well as in other factories which use large amounts of pectinolytic crude preparations, for example to enhance depuration plant performance.

Aspergillus niger↗

PubMed2002

Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up.

Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification. Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol. Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.

Bioreactors↗

PubMed2002

Composting of a crop residue through treatment with microorganisms and subsequent vermicomposting.

Preliminary studies were conducted on wheat straw to test the technical viability of an integrated system of composting, with bioinoculants and subsequent vermicomposting, to overcome the problem of lignocellulosic waste degradation, especially during the winter season. Wheat straw was pre-decomposed for 40 days by inoculating it with Pleurotus sajor-caju, Trichoderma harzianum, Aspergillus niger and Azotobacter chroococcum in different combinations. This was followed by vermicomposting for 30 days. Chemical analysis of the samples showed a significant decrease in cellulose, hemicellulose and lignin contents during pre-decomposition and vermicomposting. The N, P, K content increased significantly during pre-decomposition with bioinoculants. The best quality compost, based on chemical analysis, was prepared where the substrate was treated with all the four bioinoculants together followed by vermicomposting. Results indicated that the combination of both the systems reduced the overall time required for composting and accelerated the composting of ligno-cellulosic waste during the winter season besides producing a nutrient-enriched compost product.

Animals↗

PubMed2003

The effect of pH on the foam fractionation of beta-glucosidase and cellulase.

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.

Cellulase↗

PubMed1998

Preparation of biotinylated allosamidins with strong chitinase inhibitory activities.

NaIO4 oxidation of allosamidin (1), a strong inhibitor of family 18 chitinases, followed by a coupling with Biotin Hydrazide afforded its mono- and dibiotinylated derivatives, 4 and 6. Reduction of 4 by NaBH4 afforded its reduced form 5. Each of these three biotinylated derivatives maintained strong chitinase inhibitory activity. Especially, 6 inhibited a Trichoderma chitinase as strongly as 1.

Acetylglucosamine↗

PubMed2001

Fatty acid composition and dynamics of selected fungal-feeding nematodes and fungi.

Fatty acid profiles of fungal-feeding nematodes, Aphelenchus avenae and Aphelenchoides composticola, and selected fungi were determined in microcosm cultures of agar, broth, or sand amended with organic matter. Fatty acids of A. avenae and A. composticola included 16:0 18:0, 18:1omega7, 18:1omega9, 18:2, 20:0, 20:1, 20:2, 20:3 and 20:4 phospholipid fatty acids (PLFAs) and neutral lipid fatty acids (NLFAs). The nematodes differed in relative amounts of saturated and C(18) fatty acids. Similar C(16) and C(18) PLFAs and whole-cell fatty acids were found in Rhizoctonia solani, Fusarium oxysporum and Trichoderma sp. with 18:2omega6 as the major component. The C(20) fatty acids were not found in these fungi. Although only present in the nematodes, C(20) PLFAs were only detected when nematode population levels were > or =22 per gram of sand, suggesting that there is a detection threshold that might limit their use as biomarkers in the soil community. After removal of nematodes from a food source, the relative amount of C(20) PLFAs (structural components of nematode cell membranes) decreased more slowly than the C(16) and C(18) PLFAs, which may have reflected ingested fungal cytoplasm in the nematode intestine. In the early stage of organic matter decomposition, total and fungal PLFAs were lower in the presence of A. composticola then in its absence at C:N ratios > or =30:1.

Animals↗

PubMed2003

Interactions between nematophagous fungi and consequences for their potential as biological agents for the control of potato cyst nematodes.

The efficacies of three nematophagous fungi, Paecilomyces lilacinus, Plectosphaerella cucumerina and Pochonia chlamydosporia, for controlling potato cyst nematodes (PCN) as part of an Integrated Pest Management (IPM) regime were studied. The compatibility of the nematophagous fungi with commonly used chemical pesticides and their ability to compete with the soil fungi Rhizoctonia solani, Chaetomium globosum, Fusarium oxysporum, Penicillium bilaii and Trichoderma harzianum were tested in vitro. Paecilomyces lilacinus was the most successful competitor when the ability to grow and inhibit growth of an opposing colony at both 10 and 20 degrees C was considered. P. lilacinus also showed potential for control of the soil-borne fungal pathogen R. solani, releasing a diffusable substance in vitro which inhibited its growth and caused morphological abnormalities in its hyphae. Pochonia chlamydosporia was least susceptible to growth inhibition by other fungi at 20 degrees in vitro, but the isolate tested did not grow at 10 degrees. Plectosphaerella cucumerina was a poor saprophytic competitor. Radial growth of Paecilomyces lilacinus and Plectosphaerella cucumerina was slowed, but not prevented, when grown on potato dextrose agar incorporating the fungicides fenpiclonil and tolclofos-methyl, and was not inhibited by the addition of pencycuron or the nematicide oxamyl. Radial growth of Pochonia chlamydosporia was partially inhibited by all the chemical pesticides tested. The efficacy of Paecilomyces lilacinus as a control agent for R. solani was further investigated in situ. Treatment with P. lilacinus significantly reduced the symptoms of Rhizoctonia disease on potato stems in a pot trial. The effectiveness of P. lilacinus and P. cucumerina against PCN was also tested in situ. Three application methods were compared; incorporating the fungi into alginate pellets, Terra-Green inoculated with the fungi and applying conidia directly to the tubers. Both formulations containing P. lilacinus and formulation mixtures alone, particularly alginate pellets, significantly reduced multiplication of PCN in soil. We conclude that P. lilacinus showed the greatest potential for use in combination with selected fungicides and nematicides as part of an IPM programme for the control of PCN, but further work is required to confirm whether it is effective against PCN in soil.

Animals↗

PubMed2003

Nutritional influence on the ability of fungal mycelia to penetrate toxic metal-containing domains.

Metal-contaminated soils often contain a spatially heterogeneous distribution of metal concentrations, and the ability of fungi to colonize such metal-contaminated domains will be influenced by the nutritional resources available. An experimental system based upon tessellated agar tiles was used to study the influence of nutrients upon the ability of soil fungi Trichoderma virens and Clonostachys rosea to colonize spatially discrete toxic metal (copper and cadmium) containing domains. The growth parameters recorded demonstrated a decrease in apparent metal toxicity with increasing concentration of available carbon source. It was shown that maximum hyphal extension rates and the efficacy of carbon substrate utilization of both species decreased with increasing concentration of toxic metals. It was also observed that in the gap between metal-free and metal-containing tiles, the presence of toxic metals led to negative chemotropic reactions and cessation of growth, swelling and lysis of some hyphal tips. Penetration of the hyphae into the metal-containing domain was often followed by the formation of very dense mycelia or mycelial 'bushes' representing an associative (constraining, exploitative or 'phalanx') growth strategy of the mycelial system. After the fungi entered the toxic metal-containing domains, they often produced long sparsely-branched or branchless explorative hyphae representing a dissociative (expansive, explorative or 'guerrilla') growth strategy. Our data therefore demonstrate that fungi efficiently use both 'phalanx' and 'guerrilla' states of the mycelial system as well as shifts in these growth strategies as a response to toxic metal stress combined with nutritionally-poor conditions.

Cadmium↗