Showing papers on "Trichoderma reesei published in 1996"
TL;DR: Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei/H.
Abstract: The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.
239 citations
TL;DR: The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution, which is consistent with its proposed mode of action as a processive exoglucanase.
Abstract: Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.
198 citations
TL;DR: ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes.
Abstract: A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3 The yeast-produced enzymes were tested for enzymatic activities against different substrates ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside BXLI did not release L-arabinose from arabinoxylan It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T reesei
188 citations
TL;DR: The molecular basis of the absence of xylanase I formation on glucose was studied to postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1‐mediated carbon catabolite repression.
Abstract: The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the beta-1,4-D-xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5'-SYG-GRG-3') in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xyn1::hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xyn1 expression in a T. reesei mutant strain (RUT C-30), which contains a truncated, non-functional cre1 gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xyn1 expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression.
174 citations
TL;DR: The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively.
Abstract: The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa The construction of fur1 ura3 S cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium These autoselective S cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C
160 citations
TL;DR: Property of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers, is described.
156 citations
TL;DR: The hypothesis that Glu212 is the charged species during catalysis in cellobiohydrolase I (CBHI) from Trichoderma reesei is supported by changing these residues to their isosteric amide counterparts.
155 citations
TL;DR: It is concluded that EGI of T. reesei degradesotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.
Abstract: Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.
152 citations
TL;DR: Electrophoretic mobility shift assays, using cell-free extracts, identified induction-specific protein-DNA complexes: one complex of high mobility was observed under basal, noninduced conditions (glucose) with xyn2, which was in part replaced by a slow-migrating complex upon induction by xylan or sophorose.
132 citations
TL;DR: The solid residue of enzymatically hydrolysed SPW was found to be nearly as good a substrate as SPW, although the cellulose content was as low as 20% in this hydrolysis residue.
130 citations
TL;DR: In this paper, a description is given of the solid-state fermentation of wheat bran by Trichoderma reesei QM9414 at constant temperature and relative humidity.
Abstract: A description is given of the solid-state fermentation of wheat bran by Trichoderma reesei QM9414 at constant temperature and relative humidity. Glucosamine, the oxygen consumption rate (OCR), the carbon dioxide production rate (CPR), changes in wheat bran composition and the production of four enzymes were measured during 125 h of fermentation. A C balance was set up between CO2 production, based on CPR measurements, CO2 production as expected on the basis of substrate composition changes and substrate elemental composition in combination with dry-matter weight loss. Glucosamine was used as the measure of biomass. The results indicate that the glucosamine content of fungi in liquid culture cannot be used to estimate the biomass content in solid-state fermentations. Using glucosamine, correlations between fungal growth and respiration kinetics could only partly be described with the linear-growth model of Pirt. A decline in OCR and CPR started the moment the glucosamine level was 50% of its maximum value. After the glucosamine level had reached its maximum OCR and CPR continued to decline. The activities of xylanase and protease are linearly related to the glucosamine level. No clear correlations between glucosamine and carboxylmethylcellulose-hydrolysing enzyme activity and amylase activity were found.
TL;DR: The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin that forms SDS-insoluble complexes that could be extracted with trifluoroacetic acid.
Abstract: The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion. Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures. The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus.
TL;DR: The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme, which has a clear similarity with fungal cutinases which are serine esterases.
Abstract: The axe 1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe 1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe 1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe 1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.
TL;DR: Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter, finding the site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi.
Abstract: Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of β-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide −500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5′GTGGGG at nucleotide −720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position −161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.
TL;DR: The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the α-galactosidase previously purified from T. reesei, and the deduced amino acid sequences of A GLI and AGLIII showed similarity with the β-mannosidases of plant, animal, yeast and filamentous fungal origin.
Abstract: Three α-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevislae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. The genes agll, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the α-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLIII showed similarity with the bacterial α-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-α-d-galactopyran-oside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galac-tose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-β-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-β-mannanase of T. reesei and a β-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the α-galactosidase previously purified from T. reesei.
TL;DR: RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.
Abstract: The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.
TL;DR: The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC.
Abstract: The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4–6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1–12 s−1 at 27°C, are about 10–100 times faster than for the 4-methylumbelliferyl substituted analogs or for cellotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.
TL;DR: Data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation, and catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant.
Abstract: Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.
TL;DR: In all three complexes, clear conformational changes were found in XYNII compared to the native structure, and these changes were largest in the X-O-C3 complex structure.
Abstract: The three-dimensional structures of endo-1,4-xylanase II (XYNII) from Trichoderma reesei complexed with 4,5-epoxypentyl beta-D-xyloside (X-O-C5),3,4-epoxybutyl beta-D-xyloside (X-O-C4), and 2,3-epoxypropyl beta-D-xyloside (X-O-C3) were determined by X-ray crystallography. High-resolution measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.
TL;DR: A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei.
Abstract: The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II.
TL;DR: Results of binding assays with the maltose-binding protein::Cre1(10-131) fusion protein and the cbh1 upstream region revealed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.
Abstract: A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene (cbh1) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein: :Cre1(10–131) fusion protein (Cre1 is a catabolite repressor of T. reesei) and the cbhI upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position − 1496 to − 993) bearing three 6-bp sites, Al, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position −994 to −639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.
TL;DR: The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re‐appraised in both species.
Abstract: Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium-bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid-culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase-polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re-appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.
TL;DR: In this article, Trichoderma reesei QM 9414 was cultured to produce cellulase and biomass using as substrates untreated bagasse and bagasse alkali-treated at room temperature and at 100°C, washed with water at pH 2.
TL;DR: 1H‐NMR analysis of the action of endo‐(1 → 5)‐α‐l‐arabinanases from As pergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism, consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43.
TL;DR: Cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var.
TL;DR: A three-stagein vitromodel was used to simulate the digestion of wheat in the broiler chicken and microscopic examination of the residue showed that protein could be detected only within unbroken cells.
TL;DR: The results demonstrate that carboxyl groups are responsible for at least 30% of R. arrhizus and M. miehei zinc binding properties and strongly suggests that lipids contribu...
Abstract: Carboxyl groups contained in the cell walls of four different fungal species (Rhizopus arrhizus, Mucor miehei. Penicillium chrysogenum and Trichoderma reesei) were esterified with acidic methanol. The extent of esterification was monitored by measuring (gas chromatography) the methanol released after hydrolysis. The effect of biomass modification on metal binding was determined by zinc binding experiments at pH 5.5. A good correlation was observed between carboxyl blocking and reduction of zinc binding. Controls were also performed to examine the possible influence of methanol or acidic treatment alone. A good specificity was observed and the results demonstrate that carboxyl groups are responsible for at least 30% of R. arrhizus and M. miehei zinc binding properties. This contribution is higher with P. chrysogenum and T. reesei, with 55% and 70% of zinc binding, respectively. In addition, a non specific effect of methanol was observed with R. arrhizus, that strongly suggests that lipids contribu...
TL;DR: The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.
TL;DR: Both endoglucanase and β-glucosidase activities were found in cell wall, cell-free extracts and extramycelial fractions of Trichoderma reesei cultures grown on amorphous cellulose.
TL;DR: The cellulase system of Phanerochaete chrysosporium is adaptive and thus sensitive to carbon catabolite repression, and attempts to optimize cellulase production have led to significant improvements.