Showing papers on "Trichoderma reesei published in 1995"

Novel alkaline cellulases

Abstract: Cellulases selected from the group consisting of Family 7 cellulases and variants of these cellulases comprising a core and optionally a C-terminal link consisting of 10 amino acids at the most, especially cellulases having tryptophan, tyrosine or phenylalanine in position 55 relative to the sequence alignment of Figure 5 and/or cellulases having a substrate binding cleft of a depth of at least 12 A, exhibit enhanced enzyme activity in the alkaline pH range while exerting a moderate cellulolytic action on the cellulosic substrate and are, for example, useful in detergent compositions, especially for soil removal or colour clarification or preventing backstaining; in fabric softeners; for bio-polishing of textiles; for drainage improvement of paper pulp; for plant degradation. Cellulases from Humicola insolens, Fusarium oxysporum, Trichoderma reesei and Myceliophthora thermophile are referred to.

Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

Abstract: Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.

Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

Abstract: beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.

Cloning and amplification of the β-glucosidase gene of Trichoderma reesei

Abstract: A process for expressing extracellular β-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered β-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of β-glucosidase is also disclosed.

Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei.

Abstract: Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei. The enzyme contains a discrete cellulose-binding domain (CBD), which increases its binding and activity on crystalline cellulose. We studied cellulase-cellulose interactions using site-directed mutagenesis on the basis of the three-dimensional structure of the CBD of CBHI. Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism. The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality. The data also suggest that the more hydrophobic surface is not directly involved in the CBD function. The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption. The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect. High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD.

Cellulase production by mixed fungi in solid-substrate fermentation of bagasse.

Abstract: Ammonia-treated bagasse with 80%(w/w) moisture content was subjected to mixed-culture solid-substrate fermentation (SSF) with Trichoderma reesei LM-UC4 and Aspergillus phoenicis QM 329, in flask or pot fermenters, for cellulase production. Significantly higher activities of all the enzymes of the cellulase complex were achieved in 4 days of mixed-culture SSF than in single-culture (T. reesei) SSF. The highest filter-paper-cellulase and β-glucosidase activities seen in mixed-culture SSF were 18.7 and 38.6 IU/g dry wt, respectively, representing approx. 3- and 6-fold increases over the activities attained in single-culture SSF. The mixed-culture SSF process also converted about 46% of the cellulose and hemicellulose to reducing sugars and enriched the product with 13% fungal protein. The biomass productivity, 0.29 gl(-1).h, and enzyme productivity, 28.0 IU I(-1).h, were about twice as high in the mixed-culture than in the single-culture.

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

Abstract: A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.

The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose-inducible β-glucosidase involved in cellulase induction by sophorose

Abstract: We have investigated the effect of disruption of the bgl1-(beta-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other beta-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75 kDa extracellular beta-glucosidase on cellulose or lactose, but still formed beta-glucosidase activity on glucose, cellobiose, xylan or beta-1,3-glucan, suggesting that the enzyme(s) exhibiting this beta-glucosidase activity is (are) not encoded by bgl1. The cellulase-inducer sophorose induced the bgl1-encoded beta-glucosidase, whereas the remaining beta-glucosidase activity was induced by methyl-beta-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other beta-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase l formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The beta-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded beta-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-beta-glucoside inducible beta-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.

Production of Trichoderma reesei cellulases on glucose-containing media.

Abstract: The filamentous fungus Trichoderma reesei was shown to secrete active cellobiohydrolase I and the endoglucanase I catalytic core domain into the culture medium when the fungus was grown on glucose-containing medium. The expression of the proteins was driven by the promoters of the elongation factor 1 alpha, tef1, and the unidentified gene for cDNA1. The cDNA1 promoter gave the best yields. The highest amounts of cellobiohydrolase I and the endoglucanase I core, being 50 to 100 mg/liter, accounted for more than half of the total protein secreted by the fungus. The levels obtained with the tef1 promoter were 20 to 50 times lower.

Characterization of a Thermomonospora fusca exocellulase

Abstract: The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from Thermomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans. The E3 gene was sequenced and encoded a 596 residue peptide. The molecular masses of the native and cloned E3s were determined by mass spectrometry, and the value for E. coli E3, 59,797 Da, agreed well with that predicted from the DNA sequence, 59,646 Da. The value of 61,200 Da for T. fusca E3 is consistent with E3 being a glycoprotein. E3 is thermostable, retaining full activity after 16 h at 55 degrees C. It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10. The cloned E3s were identical to the native enzyme in their activity, cellulose binding, and thermostability. Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crystalline cellulose. E3 belongs to cellulase family B and retains the residues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T. fusca E2. The E3 gene contains a 14 bp inverted repeat regulatory sequence 212 bp before the translational start codon instead of the 30-70 bp found for the other T. fusca cellulase genes. An additional copy of this sequence with one base changed is 314 bp before the translational start codon. The transcriptional start site of the E3 gene was shown to be between these two inverted repeats.

Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation

Abstract: Growth, enzyme-producing activity and respiratory properties of Trichoderma reesei QM 9414 were examined under various agitation intensities. Two substrates were compared: lactose and Avicel. Pellet formation occurred at all agitation intensities for both substrates. Oxygen dependence at the lower agitation rate varied with the substrate type. With lactose as the carbon source, linear growth was observed, despite a regulation of the dissolved oxygen concentration at 30% saturation. The enzyme production was strongly affected by the agitation. At the higher agitation rates the enzyme production dropped. With Avicel as the carbon source, the production of enzymes surged as soon as the growth was limited by the hydrolysis of Avicel. Growth on Avicel, in the conditions we used, was limited by Avicel hydrolysis. Cubic growth was observed when lactose was the carbon source. A new derivation for a model of the observed cubic growth is proposed and is used to correlate growth, CO2 production and oxygen consumption in a consistent way, impossible with exponential growth models.

Cellulase induction by lactose in Trichoderma reesei PC-3-7

Abstract: In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005

Multiple roles of the cellulase CBHI in enhancing production of fusion antibodies by the filamentous fungus Trichoderma reesei.

Abstract: The production of Fab antibody fragments in Trichoderma reesei can be increased over 50-fold by fusing the core-linker region of the T. reesei cellulase CBHI (cellobiohydrolase I) to the heavy Fd chain (Nyyssonen et al. 1993). This beneficial role of CBHI in antibody production has now been studied further by comparisons of T. reesei strains producing the light chain only, Fab or CBHI-Fab all of which exhibited identical light chain integration. The N-terminal fusion of CBHI to the heavy Fd chain not only aided secretion, as expected, but also increased the level of mRNA encoding the CBHI-heavy Fd chain, either by stabilizing the messenger or by enhancing transcription. The CBHI part appeared to facilitate secretion at least by aiding the passage through the endoplasmic reticulum, since processing of the signal peptide of the antibody chains seemed to be most efficient in the strain producing CBHI-Fab in contrast to the strains producing light chain or Fab fragment. Interestingly, CBHI core-linker protein, originating from the CBHI-heavy Fd chain, was found in large amounts in the culture medium. The cleavage resulting in this tailless CBHI occurred inside the cell. This suggests that, by omitting the heterologous tail, the secretion of the resulting CBHI core-linker protein is enhanced to a level comparable with secretion of the extracellular T. reesei proteins.

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract: Eleven strains of Trichoderma viride, 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica, using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum, a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.

Molecular Biology of Cellulolytic Fungi

Abstract: The synthesis, modification and hydrolysis of carbohydrates by glycosidase enzymes are some of the fundamental activities in nature. Enzymes responsible for these processes are produced across different organisms, genera and species including the kingdom fungi. Together with bacteria, fungi are responsible for the recycling of nature’s recalcitrant polymers such as lignocellulose which is mainly stored in the plant cell walls. The three main components of a plant cell wall are cellulose, hemicellulose and lignin in a percent ratio of about 40:30:30 (Sjostrom 1981). White rot fungi are capable of degrading all three polymeric substances, including the polyphenolic lignin, whereas brown rot and soft rot fungi prefer the carbohydrate polymers of cellulose, formed of 13-1,4-linked D-glucopyranose units with no side branches and hemicellulose, of which the backbone structure consists of s-1,4-linked D-xylopyranosyl units (xylan) or 13-1,4-linked D-mannose and D-glucose units (mannan) with sugar side chains that may be acetylated and/or methylated (reviewed in Tenkanen 1995). Earlier studies of lignocellulose hydrolysis have mainly concentrated on the biochemistry and molecular biology of cellulose degradation. More recently, the enzymology of lignin degradation (reviewed in Leonowicz et al. 1999) and especially molecular studies on the hydrolysis of hemicellulose have advanced considerably (e.g. de Vries et al. 2002). Xylan degradation has been studied in detail with genes and enzymes from Aspergillus (reviewed in de Vries et al. 2002) and lignin degradation with Phanerochaete chrysosporium (reviewed in Cameron et al. 2000). At present, some 20 enzymes involved in the degradation of lignocellulose have been described. In this chapter, we will concentrate on molecular aspects relating to cellulose hydrolysis.

Kinetic and stereochemical studies of manno-oligosaccharide hydrolysis catalysed by beta-mannanases from Trichoderma reesei.

Abstract: The two β-mannanases from Trichoderma reesei with pI of 4.6 and 5.4, respectively, have been characterised by NMR spectroscopy. Following the kinetics of manno-oligosaccharide degradation with complete progress-curve analysis the stereospecificity and degradation pattern have been delineated. It was found that degradation of mannotriose and mannopentaose proceeds with retention of the anomeric configuration. Mannotriose degradation proceeds by almost random release of mannose. For mannopentaose there is initially no mannose formed showing that only the two middle mannosidic linkages are attacked. Progress-curve analysis shows that there is preference (70%) for cleavage of mannopentaose in such a way that mannobiose is released from the reducing end. The final product composition from the mannotriose degradation showed that transglycosylation has to be taken into account. Model calculation and progress-curve analysis showed that the transglycosylation rate is the fastest of all the rates in this system, 15 s-1 compared with mannohexaose and mannotetraose hydrolysis rates of 2 s-1 and mannotriose hydrolysis rate of 0.03 s-1 at 50°C.

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger, Neurospora crassa, Penicillium chrysogenum and Trichoderma reesei)

Abstract: The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi:Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, andTrichoderma reesei. Fatty acid unsaturation increased inA. niger, P. chrysogenum, andT. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. InA. niger andT. reesei, this was due to the increase in linolenic acid content. InP. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. InT. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. InA. niger andP. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, inN. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses.

Mitochondrial functions mediate cellulase gene expression in trichoderma reesei

Abstract: We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.