Showing papers on "Trichoderma reesei published in 1983"
TL;DR: The molecular cloning and characterization of the gene encoding exo–cellobiohydrolase I of Trichoderma reesei strain L27 is reported and it is found to contain a 10 bp sequence, CAGCT–GACTG, that is homologous to a sequence necessary for splicing of introns in yeast2.
Abstract: The molecular cloning and characterization of the gene encoding exo–cellobiohydrolase I (CBHI) of Trichoderma reesei strain L27 is reported. Two adjacent HindIII genomic fragments of 1.16 kb and 2.3 kb were identified using differential hybridization techniques and were sub–cloned into plasmid pBR322. The identification of the gene encoding CBHI was determined by hybrid selection and confirmed by DNA sequence analysis. There are two introns in the genomic DNA that were identified by comparing the coding sequence with the published amino acid sequence1 and confirmed by sequencing of cDNA clones. Both introns were found to contain a 10 bp sequence, CAGCT–GACTG, that is homologous to a sequence necessary for splicing of introns in yeast2.
408 citations
TL;DR: It is shown by hybrid messenger selection and partial DNA sequencing that one of the clones contains a gene coding for the major cellulolytic enzyme of T. reesei, cellobiohydrolase I.
Abstract: The filamentous fungus Trichoderma reesei produces a number of inducible cellulolytic enzymes. By differential hybridization of cDNA probes made from induced and repressed messenger RNA populations to a λ phage gene bank, we have isolated a number of genes strongly expressed during production of cellulolytic enzymes. We have shown by hybrid messenger selection and partial DNA sequencing that one of the clones contains a gene coding for the major cellulolytic enzyme of T. reesei, cellobiohydrolase I.
200 citations
TL;DR: The ‐;xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of β‐glucosid enzyme in the saccharification of cellulose by cellulases.
Abstract: The growth of Trichoderma reesei QM9414 in shake flasks at 28 degrees C on hemicellulose substrates and bagasse resulted in rather low yields of hemicellulolytic enzymes (1.0-1.5 units/mL xylanase and 0.05-0.08 units/mL beta-xylosidase). The influence of pH on the synthesis of beta-xylosidase was greater than on the synthesis of xylanase. Both xylanase and beta-xylosidase showed optimal activity at pH 4-5 and 55-60 degrees C. Xylanase was stable at pH 2-10 but was heat labile and totally inactivated after 1 h at 65 degrees C. Enzyme stability towards heat could be increased in the presence of bovine serum albumin. The beta-xylosidase was more tolerant to heat, but stable over a pH range 2.5-6.0. The D-xylose inhibited both enzymes in a competitive manner. Hemicellulose (heteroxylan) was degraded to the extent of 30-40%within 24 h. The degree of hydrolysis decreased as the substrate concentration increased and increased with increased amounts of enzyme. Multiple enzyme doses resulted in increased saccharification in reduced times. The degree of hydrolysis was influenced by the amount of beta-xylosidase present in the hemicellulolytic enzyme preparation. The -;xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of beta-glucosidase in the saccharification of cellulose by cellulases.
158 citations
TL;DR: The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, and the result has shown that the action of β‐glucosidase is competitively inhibited by glucose.
Abstract: The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.
146 citations
TL;DR: In this paper, the authors showed that the growth improvement of chicks fed enzyme-supplemented barley diets could not be due to improved glucose availability from β-D-glucan.
137 citations
TL;DR: Progress is being made in the delineation of the biochemical mechanisms of cellulose hydrolysis, strain improvement, molecular cloning and process engineering that are bringing T. reesei cellulases closer to being a commercially viable route to cellulose Hydrolysis.
137 citations
TL;DR: Amino terminal protein sequencing of the first 30 amino acid residues of CBHI and EGI indicates significant homology, however, high pressure liquid chromatography analysis of tryptic peptides revealed little or no homology between these two proteins.
Abstract: Trichoderma reesei strain L27 produces multiple 1,4–β–D–glucan cellobiohydrolases, endo–1,4–β–D–glucanases, and 1,4–β–D–glucosidases. The major cellobiohydrolase (CBHI) and endoglucanase (EGI) have been purified to homogeneity and a β–glucosidase (BGI) has been substantially purified. Specific antibodies have been raised against these proteins. Amino terminal protein sequencing of the first 30 amino acid residues of CBHI and EGI indicates significant homology. High pressure liquid chromatography (HPLC) analysis of tryptic peptides, however, revealed little or no homology between these two proteins.
135 citations
TL;DR: Factors which influenced the extent of saccharification of autohydrolysis‐exploded bagasse pulps were the enzyme‐substrate ratio, the substrate concentration, and the saccharifications mode.
Abstract: Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a comercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogenous beta-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lowere conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h) both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulose preparations with A. niger beta-glucosidases was effective in promoting the conversion of cellulose into glucose. A ration of FPU to beta-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification of autohydrolysis-exploded bagasse pulps were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode.
119 citations
TL;DR: The interaction between 1,4-β-Dglucan-cellobiohydrolase I (CBHI) from Trichoderma reesei and microcrystalline cellulose from Vallonia macrophysa was investigated by electron microscopy.
105 citations
TL;DR: A peptide antibiotic has been isolated from Trichoderma reesei QM 9414 as discussed by the authors, which is crystalline and uniform in TLC, this antibiotic could be resolved by HPLC into 3 sequence analogues.
Abstract: A peptide antibiotic has been isolated from Trichoderma reesei QM 9414. Although crystalline and uniform in TLC, this antibiotic could be resolved by HPLC into 3 sequence analogues. The close relationship to alamethicin was proved by chemical and spectroscopic methods, and the formation of ion-conducting pores in lipid bilayers.
98 citations
TL;DR: A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography.
Abstract: A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.
TL;DR: It is concluded that the presence of multiple enzyme forms is an inherent property of the cellulase system of Trichoderma and cannot be considered solely as the result of proteolytic postsecretional modification of secreted extracellular enzymes.
TL;DR: The action of a commercial cellulase preparation from Trichoderma reesei on microcrystalline cellulose is compared with that of cellulolytic enzymes from other microorganisms and it is concluded that the Trichodma system is not unique.
Abstract: Several processes have been developed for the enzymic conversion of lignocellulose to fermentable sugars. Most of these processes have employed the cellulolytic enzyme system from Trichoderma reesei. The action of a commercial cellulase preparation, Celluclast, from Trichoderma reesei on microcrystalline cellulose is compared with that of cellulolytic enzymes from other microorganisms. It is concluded that the Trichoderma system is not unique. More effective enzyme complexes can be produced from other microorganisms. In particular, systems from different Aspergillus species are shown to be more effective. The enzymic conversion of lignocellulose to fermentable sugar has not yet been scaled up, undoubtedly for technical and economic reasons. The cellulose substrate is very inaccessible, and furthermore the enzymic hydrolysis involves several consecutive reactions, each of which may be rate limiting. Enzyme costs have a great impact on process economy. The conversion of cellulose into fermentable sugar requires approximately 100 times more enzyme protein than the corresponding hydrolysis of gelatinized starch. Therefore, with today's enzyme technology, the use of cellulose as a raw material is not competitive with processes based on starch.
TL;DR: Culture filtrates of CL‐847 strain of Trichoderma reesei grown on different carbon sources have been compared and about one‐third of maximal enzyme production is obtained on lactose as the sole carbon source.
Abstract: Culture filtrates of CL-847 strain of Trichoderma reesei grown on different carbon sources have been compared The highest enzyme production is obtained with Whatman C 41 cellulose: 179 mg/mL of soluble proteins and 137 units of filter paper (FP) activity Wood pulps gave lower production values and more viscous culture media About one-third of maximal enzyme production is obtained on lactose as the sole carbon source Addition of 05% cellulosic inducer to 6% lactose media enhances enzyme production up to the following levels: 141 mg/mL of soluble proteins and 84 units of FP activity
01 Jan 1983
TL;DR: A model is proposed, which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate of Trichoderma reesei, which increases the time to attain a steady state to 4–6 weeks in a two-stage system.
Abstract: A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h−1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h−1 all of these properties change due to derepression (for D 0.025 h−1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.
TL;DR: In this article, the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants were reported.
TL;DR: The ability of each enzyme system, separately and together, to digest beet pulp was investigated and the cellulase system from Trichoderma reesei was decidedly less stable than that of Talaromyces emersonii under each of the above conditions.
TL;DR: In this article, the enzymic degradation of cellulose from Valonia macrophysa was observed by electron microscopy and evaluated by electron diffraction, and two types of Valonia samples were subjected to digestion; first, intact fragments cut from cell wall material and, secondly, cellulose microcrystals resulting from the acid hydrolysis of entire vesicles.
TL;DR: The β-glucanase in commercial |cellulases prepared from Penicillium funiculosum is more stable to heating than is the equivalent enzyme system from Trichoderma reesei, and it follows that this enzyme is to be preferred for use in the enzymic method for measuring β- glucan.
Abstract: The β-glucanase in commercial |cellulases prepared from Penicillium funiculosum is more stable to heating than is the equivalent enzyme system from Trichoderma reesei. Consequently the heat-labile amyloglucosidase can be destroyed more reliably in Penicillium cellulase, and it follows that this enzyme is to be preferred for use in the enzymic method for measuring β-glucan.
TL;DR: In this article, the authors used enzymes to extract sugar from sunflower seed hulls at 200°C for 5 min followed by explosive defibration solubilized >80% of the total hemicellulose (heteroxylan) and 85% of pectic substances, which consisted of cellulose (38%), lignin (45%), and residual heteroxylan (7%).
Abstract: Autohydrolysis of sunflower seed hulls at 200°C for 5 min followed by explosive defibration solubilized >80% of the total hemicellulose (heteroxylan) and 85% of the pectic substances The remaining residue, which consisted of cellulose (38%), lignin (45%) and residual heteroxylan (7%), was highly susceptible to hydrolysis by cellulases (Trichoderma reesei C-30, Meicelase and Onozuka 3S) Enzymic digests containing 10% (w/v) substrate and 20 FPU/g pretreated hull resulted in sugar yields of >40% within 24h This yield was increased toca 70% when digests were supplemented with exogenous β-glucosidase The method has potential use in the bioconversion of sunflower seed hulls into chemicals
01 Sep 1983
TL;DR: The proteins in the supernatant of Trichoderma reesei were separated by HPLC and exo-, endo-β 1,4-glucanase, and β- glucosidase activities for the various fractions obtained were measured.
Abstract: The proteins in the supernatant of Trichoderma reesei were separated by HPLC and exo-, endo-β 1,4-glucanase, and β-glucosidase activities for the various fractions obtained were measured. Bovine serum albumin (BSA), chymotrypsin, β-glucosidase, endoglucanase and other cellulase preparates were used as reference substrates.
TL;DR: For enhanced production of cellulase and xylanase by the mixed culture of T. reesei D1-6 and A. wentii Pt 2804, the composition of medium has been optimized.
Abstract: For enhanced production of cellulase and xylanase by the mixed culture ofT. reesei D1-6 andA. wentii Pt 2804, the composition of medium has been optimized.
Journal Article•
TL;DR: The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% 2-deoxyglucose.
Abstract: The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.
TL;DR: The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as theCatabolite repressor.
Abstract: The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.
01 Mar 1983
TL;DR: Sphaeroplasts from several genetically marked strains of Trichoderma reesei were readily prepared through enzymatic hydrolysis of mycelial suspensions utilizing Driselase, a commercially available lytic enzyme preparation, resulting in increased sphaeroplast yields from young as well as older mycelia.
Abstract: Sphaeroplasts from several genetically marked strains of Trichoderma reesei were readily prepared through enzymatic hydrolysis of mycelial suspensions utilizing Driselase, a commercially available lytic enzyme preparation. Sphaeroplasts were released from the apical tips of hyphae after 90 min exposure to the enzyme and, with longer treatments, from other regions of the hyphae. The efficiency of sphaeroplast formation was dependent upon a number of factors. Young mycelium was far more susceptible to lysis than older hyphae. Additionally, the yields of sphaeroplasts were directly proportional to the mycelial concentration within the range tested (0.7–7.0 mg dry weight of mycelium). Ammonium sulfate (0.6 M) was the most effective osmotic support in relation to sphaeroplast stability during their release from the mycelium and subsequent purification. The most prominent enzymatic activities of Driselase were endo-β-(1→4)-glucanase, β-glucosidase and β-(1→3)-glucanase. No chitinase activity was detected. The combined application of Driselase and chitinase (Streptomyces griseus) to mycelial suspensions resulted in increased sphaeroplast yields from young (18 h) as well as older (24 h) mycelia. The parameters defined here allow the rapid (<6h), inexpensive production of 107 sphaeroplasts/ml. Regeneration of T. reesei sphaeroplasts proceeded by the production of chains of sphaeroplast-like cells followed by true hyphal formation. The frequency of regeneration to mycelial form was dependent upon the length of exposure of mycelium to the lytic enzyme. Less than 1% of the sphaeroplasts prepared from 24 h hydrolytic treatment of mycelia regenerate to form colonies while virtually 100% of the viable sphaeroplasts prepared from 6 h treatment form colonies in regeneration medium.
TL;DR: The cellulase was used to hydrolyze cellulose (spruce sulfite pulp) and various lignocellulosic waste materials (newspaper, wheatstraw, cornstover, wood and reed grass a.o.).
Abstract: Cellulase was produced by cultivating T. reesei QM 9414 and MCG 77 on spruce sulfite pulp. The solid enzyme prepared by acetone precipitation had 0.73 FPU/mg, 0.2 units s-glucosidase/mg and 10 units xylanase/mg. The average molecular weight was 48000 dalton. Analytical and preparative (20 mg) separation of the cellulase by chromatofocusing revealed the presence of 18 different proteins. Identified were: two exocellulases (C1 -enzymes), five endocellulases (Cx -enzymes), one s-glucosidase, one galacto-mannase, two xylanases and one s-xylosidase. The cellulase was used to hydrolyze cellulose (spruce sulfite pulp) and various lignocellulosic waste materials (newspaper, wheatstraw, cornstover, wood and reed grass a.o.). With 10, 5, 2.5, 1 and 0.5% cellulose slurries percentage of saccharification (24 hours) was 25, 42, 45, 53 and 69 %. Saccharification increased with increasing enzyme concentration up to a level of 0.60 FPU/ml and 0.36 units s-glucosidase/ml. A further increase of either one of the e...
TL;DR: Production of cellulases by three different genetically improved strains of Trichoderma reesei: MCG 77, RUT-C30 and CL-847, has been assessed on various fermentation media.
Abstract: Production of cellulases by three different genetically improved strains of Trichoderma reesei: MCG 77, RUT-C30 and CL-847, has been assessed on various fermentation media. The three strains produce high levels of enzymes when grown on purified cellulose as the main carbon energy source; when grown on lactose, decrease in enzyme yield and productivity, differs significantly from strain to strain.
TL;DR: It is shown that the nature of both the enzymes and the substrate make the assay unsuitable for use in the calculation of enzyme recovery and purity.
Abstract: A purified endocellulase from Sclerotium rolfsii and a crude cellulase preparation from Trichoderma reesei are used to illustrate several pitfalls associated with the assay of carboxymethylcellulase activity and the subsequent attainment of linear enzyme dilution curves. It is shown that the nature of both the enzymes and the substrate make the assay unsuitable for use in the calculation of enzyme recovery and purity.
TL;DR: The results indicate the potential of this strain of Penicillium funiculosum as an attractive alternative to Trichoderma reesei and its mutants.