Showing papers on "Trichoderma reesei published in 1991"

An enzyme capable of degrading cellulose or hemicellulose.

Abstract: A cellulose- or hemicellulose-degrading enzyme which is derivable from a fungus other than Trichoderma or Phanerochaete, and which comprises a carbohydrate binding domain homologous to a terminal A region of Trichoderma reesei cellulases, which carbohydrate binding domain comprises amino acid sequence (α) or a subsequence thereof capable of effecting binding of the enzyme to an insoluble cellulosic or hemicellulosic substrate.

Thermostable enzymes for industrial applications

Abstract: The variety of thermostable (TS) enzymes has been steadily increasing for use in industrial applications, mainly as replacements for thermolabile (TL) enzymes. For example, TS amylases fromBacillus licheniformis andBacillus stearothermophilus have replaced TL amylases fromBacillus subtilis. TS enzymes also have advantages in new areas such as cyclodextrin production. The TS cyclodextrin glycosyl transferase (CGTase) fromThermoanaerobacter sp. (95°C optimum) gives a higher productivity than the CGTase fromBacillus macerans (55°C optimum). In the area of enzymatic bleach boosting of wood pulps, a TS xylanase (Myceliophera thermophila) would be advantageous over a TL xylanase (Trichoderma reesei), due to the high temperature of the incoming pulp. Not all TS enzymes are from thermophiles; the mesophileCandida antarctica produces a TS lipase which has a temperature optimum of 90°C when immobilized. The characterization of these enzymes will be described along with comparisons to some newly described TS enzymes.

Dilute-Acid Pretreatment of Corn Residues and Short-Rotation Woody Crops

Abstract: As a prerequisite for the enzymatic saccharification or simultaneous saccharification and fermentation process for ethanol synthesis, a dilute-acid pretreatment of the biomass has been shown to be a very effective first step in the yeast-catalyzed bioprocess. Three hardwoods (silver maple, sycamore, and black locust) and two corn residues (cobs and stover) were chosen and subjected to prehydrolysis with dilute sulfuric acid at 140 and 160 °C for reaction times ranging from 5 to 60 min. Although the hemicelluloses from all five samples could be completely hydrolyzed at both 140 and 160 °C, hydrolysis at 160 °C for the woods and stover produced a superior substrate for the cellulase enzyme from Trichoderma reesei, in which > 90% of the cellulose was hydrolyzed by the enzyme. Corn cobs produced an excellent substrate after only 5 min at 140°C. Small amounts of lignin and glucan were also solubilized by the acid in all samples.

Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

Abstract: We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

Saccharification of steam‐exploded poplar wood

Abstract: Effects of time, temperature, and pH during the steam explosion of poplar wood were studied with the aim of optimize both pentoses recovery and enzymatic hydrolysis efficiency. Steam explosion of acid impregnated wood chips allowed the recovery of 70% of potential xylose as monomers (217 degrees C, 120 s) Enzymatic hydrolysis of pretreated fiber with Trichoderma reesei CL-847 cellulase system increased progressively with the severity of the steam treatment conditions. The best yield in term of glucose recovery after 24 h of enzymatic hydrolysis was 70% of potential glucose (225 degrees C, 120 s). Deactivation by adsorption on lignin of Trichoderma reesei cellulases and inhibition of these enzymes by low-molecular-weight phenols and trihydroxybutyric acids were noticed.

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

Abstract: The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon There were no significant homologies between the coding regions of the three genes The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes E2 belongs to a family of cellulases that includes celA from M bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C fimi and Pseudomonas fluorescens cellulose binding domains

Purification and characterization of an antifungal chitinase from Arabidopsis thaliana

Abstract: Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907-914).

Heterologous Production of a Ligninolytic Enzyme: Expression of the Phlebia Radiata Laccase Gene in Trichoderma Reesei

Abstract: We have expressed a gene encoding the ligninolytic laccase enzyme of the white–rot fungus Phlebia radiata in the soft rot fungus Trichoderma reesei under the promoter of the major cellulase gene (cbh1). Constructions from the laccase cDNA or the chromosomal gene were equivalent in expression, yielding 20 mg/l of secreted active laccase in small–scale fermentations. The recombinant enzyme has a similar molecular weight, antigenic properties and specific activity as the Phlebia laccase and is analogous to the Phlebia enzyme in removing monomeric lignin–related compounds from residual lignin of the pulping process, thus demonstrating its potential for industrial applications.

The endo-1,4-beta-glucanase I from Trichoderma reesei. Action on beta-1,4-oligomers and polymers derived from D-glucose and D-xylose

Abstract: The reaction mechanism of the non-specific endo-1,4-beta-glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing-end3H-labelled and universally 14C-labelled cellooligosaccharides, as well as reducing-end3H-labelled xylooligosaccharides. The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible. During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive D-glucose or cellobiose. The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize cellooligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of D-glucosyl and D-xylosyl residues. The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, -II and +II show the highest affinities, 16.7 kJ.mol-1 and 7.1 kJ.mol-1, respectively.

Enzymes in biomass conversion.

Abstract: Enzymes for Fuels and Chemical Feedstocks Enzymes in Pulp and Paper Processing Enzymes for Anaerobic Municipal Solid Waste Disposal Thermostable Saccharidases: New Sources, Uses, and Biodesigns Alkali-Enzymes Produced by an Alkaliphilic Bacillus Strain: Mannan-Degrading Enzymes Produced by Bacillus sp. AM-001 Proteinases and Their Inhibitors in Biotechnology Subtilisin: Commercially Relevant Model for Large-Scale Enzyme Production Enzymes from Solid Substrates: Recovering Extracellular Degradative Enzymes from Lenticula edodes Cultures Grown on Commercial Wood Medium Production of Trichoderma reesei Cellulase System with High Hydrolytic Potential by Solid-State Fermentation Role of Statistically Designed Experiments in the Development of Efficient Downstream Processes Enhanced Utility of Polysaccharidases through Chemical Cross-linking and Immobilization Applications Bioprocessing Aids in the Recovery of Proteins from Biomass Chromatography in Enzyme Isolation and Production Lignin Peroxidase: Catalysis, Oxycomplex, and Heme-Linked Ionization Structure and Regulation of Manganese Peroxidase Gene from Phanerochaete chrysosporium Regulation of Ligninase Production in White-Rot Fungi Laccases of the Ligninolytic Fungus Pilot-Scale Production and Properties of Lignin Peroxidases Chemistry of Lignin Degradation: An Extracurricular View Lignin-Carbohydrate Complexes from Polar Wood: Isolation and Enzymatic Degradation Cellulase: Insights through Recombinant DNA Approaches Structure of Cellulolytic Enzymes Thermal Unfolding of Trichoderma reesei CBH I Bacterial Cellulases Regulation of Synthesis Cellulomonas fimi *b-1,4-Glucanases Comparison of Amylopullulanase to *a-Amylase and Pullulanase Cyclodextrin Glucanotransferases: Technology and Biocatalysis Design Starch Liquefacation with a Highly Thermostable Cyclodextrin Glycosyl Transferase from Thermoanaerobacter sp. Reactions of Glucansucrases in the Biomass Conversion of Sucrose Biotechnological Potential and Production of Xylanolytic Systems Free of Cellulases Catalytic Properties and Partial Amino Acid Sequence of an Actinomycete Endo-(1RT4)-*b-D-Xylanase from Chainia sp. Accessory Enzymes Involved in the Hydrolysis of Xylans Comparison of Endolytic Hydrolases That Depolymerize 1,4-*b-D-Mannan, 1,5-*a-L-Arabinan, and 1,4-*b-D-Galactan Microbial Strategies for the Depolymerization of Plant and Algal Polyuronates Synergism between 1,3-*b-Glucanases in Yeast Cell Wall Zymolysis Chitinases

Trichoderma reesei containing deleted and/or enriched cellulase and other enzyme genes and cellulase compositions derived therefrom

Abstract: A process for transforming the filamentous fungus T. reesei which involves the steps of treating a T. reesei strain with substantially homologous linear recombinant DNA to permit homologous transformation and then selecting the resulting T. reesei transformants. Transformants made by this process are disclosed, as well as cellulase compositions prepared via the transformed strains.

Purification and properties of two acetylxylan esterases of Trichoderma reesei

Abstract: Two acetylxylan esterases (AXE I and AXE II) of Trichoderma reesei were purified to homogenity using ion-exchange and gel chromatography and chromatofocusing. The two isoenzymes had very similar properties. The isoelectric point of AXE I was 7.0 and that of AXE II 6.8. Both enzymes were monomeric glycoproteins having a relative molecular mass of 34,000 as determined by sodium dodecyl sulfate electrophoresis or 20,000 as determined by gel filtration. They were markedly stable, retaining full activity at 45 o C for 24 h at pH values between 3 and 7 and up to 60 o C at pH 5.0. The purified enzymes were alone capable of liberating acetic acid from acetylated xylo-oligomers. They also hydrolyzed acetylated monosaccharides and aromaticd acetates, but not aliphatic acetates

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Abstract: Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei.

Abstract: Regulation of the formation and secretion of two cellulase components from Trichoderma reesei QM 9414, cellobiohydrolases I and II (CBH I and CBH II, respectively), by the carbon source was investigated. With monoclonal antibodies against CBH I and CBH II it was found that during cultivation on carbon sources which enable fast growth (glucose, glycerol, and fructose), no formation of CBH I occurred, whereas low levels of CBH II were formed. Lactose and cellulose, which allow comparably slower growth, promoted the formation of both CBH I and CBH II. However, noncarbohydrate carbon sources as citrate or acetate, which also enable only slow growth, did not promote the formation of CBH I or CBH II. The addition of glucose or glycerol to lactose- or cellulose-pregrown mycelia, on the other hand, only partially reduced the formation of CBH I. This reduction was also achieved by several other metabolizable and nonmetabolizable carbon compounds, e.g., fructose, galactose, beta-methylglucoside, 2-deoxyglucose, and rhamnose, as well as by transfer to no carbon source at all. This result indicates that the control of CBH I synthesis by the carbon source is due to induction and not to repression. The use of cycloheximide and 5-fluorouracil as inhibitors at and before translation, respectively, revealed a half-life for CBH I mRNA of at least several hours, which may, at least in part, account for the prolonged synthesis of some CBH I under these conditions. Northern (RNA) hybridization with full copies of cbh1 and cbh2 genes indicated that the control of CBH I and CBH II biosyntheses by the carbon source operates mainly at the pretranslational level. We conclude that the low rate of cellulase synthesis on glucose and some other carbon sources is due to the lack of an inducer and not to carbon source repression.

Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes

Abstract: Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5′-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (>104 transformants per μg DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Abstract: Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and β-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Enzyme capable of degrading cellulose or hemicellulose

Abstract: A cellulose- or hemicellulose-degrading enzyme which is derivable from a fungus other than Trichoderma or Phanero-chaete, and which comprises a carbohydrate binding domain homologous to a terminal A region of Trichoderma reesei cellulases, which carbohydrate binding domain comprises amino acid sequence (.alpha.) or a subsequence thereof capable of effecting binding of the enzyme to an insoluble cellulosic or hemicellulosic substrate.

Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation.

Abstract: We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5′-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.

Improved saccharification of cellulose by cloning and amplification of the beta-glucosidase gene of trichoderma reesei

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

Cellulase production by Trichoderma reesei when cultured on xylose‐based media supplemented with sorbose

Abstract: The ability of L-sorbose to stimulate cellulase production In shake flask culture of Trichoderma reesei was examined in mineral salts media (initial pH 5.0) containing either 1.0% D-xylose, 1.0% cellulose, and/or 0.1, 0.3, or 0.5% L-sorbose. When sorbose was the only carbon source, growth was limited, little substrate was utilized, pH increased, and cellulase activity was not apparent. The other carbon sources promoted good growth, pH dropped sharply to 2.5–3.0, substrate was utilized rapidly, and cellulase activity was detected. After three weeks of fermentation, twice as much cellulase activity was detected in the medium containing only cellulose as the carbon source, as compared to xylose as the carbon source. Cellulase activity was higher when media contained xylose supplemented with sorbose compared to xylose as the only carbon source. At 0.3 and 0.5% levels of sorbose supplementation of xylose-based media, cellulase activity was similar to that in cellulose-based media.