Showing papers on "Trichoderma reesei published in 2001"

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

Abstract: There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM.

Abstract: A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

Abstract: Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to “fingerprint” analysis by high-resolution 2-D gel electro-phoresis. Forty-five spots from 11 × 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of compo-nent enzymes from the T. reesei cellulase system acting on complex sub-strates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggestfollow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Degradation of Xylan to D-Xylose by Recombinant Saccharomyces cerevisiae Coexpressing the Aspergillus niger β-Xylosidase (xlnD) and the Trichoderma reesei Xylanase II (xyn2) Genes

Abstract: Plant cell walls, the major reservoir of fixed carbon in nature, contain three major polymers: cellulose (insoluble fibers of β-1,4-glucan), hemicellulose (noncellulosic polysaccharides including xylans, mannans, and glucans) and lignin (a complex polyphenolic structure) (1, 45). β-1,4-Xylans are found mainly in secondary walls of plants and can represent up to 35% of the total dry weight in certain plants. Xylan is a complex polysaccharide consisting of a backbone of β-d-1,4-linked xylopyranoside units substituted with acetyl, glucuronosyl, and arabinosyl side chains. Endo-β-xylanases (EC 3.2.1.8) act on xylans and xylo-oligosaccharides, producing mainly mixtures of xylooligosaccharides (4, 23). β-d-Xylosidases (EC 3.2.1.37) hydrolyze xylooligosaccharides, produced through the action of β-xylanases, to d-xylose. Many bacterial and fungal species are able to utilize xylans as a carbon source (18). Strains of the fungi Trichoderma and Aspergillus secrete large amounts of efficient xylan-degrading enzymes (8, 16, 51). Recently, interest in β-xylanases has increased because of their application in biobleaching (30, 44) and the food (31) and animal feed (3, 34, 47) industry. Trichoderma reesei is a filamentous mesophilic fungus that is well known for its cellulolytic and xylanolytic enzymatic activities (12, 43). The two major inducible endo-β-xylanases secreted by this fungus are Xyn1 and Xyn2 (46). They are both relatively small protein molecules, with molecular masses of 19 and 21 kDa, respectively, but Xyn2 represents more than 50% of the total xylanolytic activity of T. reesei cultivated on xylan. Fungi of the genus Aspergillus are also efficient producers of cellulose- and xylan-degrading enzymes, regulated at the transcriptional level by the XlnR activator (49). The two endo-β-xylanases and the β-xylosidase in A. niger are encoded by xlnB, xlnC, and xlnD, respectively. The xlnD gene contains an open reading frame of 2,412 nucleotides, which encodes a protein of 804 amino acids with a predicted molecular mass of 85 kDa. The protein is N glycosylated and contains 15 potential N-glycosylation sites (48). Sequence similarity was found to β-glycosidases (β-xylosidase and β-glucosidases) of family 3, which include enzymes from both bacterial and fungal origins (20, 33, 35, 48). The condensation reaction of this β-xylosidase has been used for the synthesis of disaccharides such as β,β-1,1-xylodisaccharide, β-1,4-xylodisaccharide (xylobiose), β-1,2-xylodisaccharide, α-1,4-xylodisaccharide, and β-1,3-xylodisaccharide (17). S. cerevisiae has been successfully used for the production of related fungal β-xylosidase and β-glucosidases belonging to family 3 (7, 32, 33). Different Candida species (C. maltosa, C. tropicalis, and C. utilis) are currently used in industry for the production of single-cell protein and ethanol from steamed hemicellulose (21). Even though these Candida strains are able to ferment d-xylose, none of them are able to tolerate the same levels of ethanol as Saccharomyces cerevisiae does, and, furthermore, they cannot ferment hexose sugars as effectively. However, the main disadvantage of S. cerevisiae is the fact that it cannot hydrolyze xylan or utilize or ferment d-xylose, the main component of xylan. While research is continuing on the development of a S. cerevisiae strain able to ferment d-xylose (9, 14, 28, 50), we are working toward the construction of strains able to break down the xylan backbone to its monomeric constituent, d-xylose. In this paper, we describe the molecular cloning of the A. niger xlnD gene and its expression in S. cerevisiae. Expression and coexpression of xlnD and xyn2 from T. reesei in yeast was obtained with the aid of multicopy plasmids using the derepressible S. cerevisiae alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences (38). The enhanced production of both the recombinant enzymes in non-selective complex medium, without the risk of losing the episomal vector, was obtained by constructing autoselective recombinant fur1 S. cerevisiae strains (29).

Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI

Abstract: Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l(-1)) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.

Pilot-scale production of cellulase using Trichoderma reesei Rut C-30 in Fed-Batch mode

Abstract: Trichoderma reesei Rut C-30 produced high levels of -glucosidase, endo--glucosidase, endo--1,4-glucanase, and exo--1,4-glucanase. In pilot-scale production (50-1 fermentor), productivity and yield of CMCase (carborymethyl cellulose) and FPase (filter paper activity) were 273 U/ml and 35 U/ml, and 162 FPU/l.h and 437 FPU/g, respectively. The fed-batch techniques were used to improve enzyme activities with constant cell concentration. The acidity was an important parameter and controlled at pH 3.9 and 5.0 by automatic addition of ammonium hydroxide. Cellulase powder was prepared by ammonium sulfate precipitation and its CMCase and FPase activities were 3,631 U/g and 407 U/g, respectively.

L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei.

Abstract: l-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, l-sorbose was found to regulate coordinately six cellulase genes (including egl3, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when l-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45–60% and 20–30% of the cbh1 transcript, whereas small amounts of mRNA, 1–2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and l-sorbose, respectively.

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium

Abstract: Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant.

Abstract: The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum

Abstract: Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach.

Abstract: A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

Cellulose-binding domain of endoglucanase III from Trichoderma reesei disrupting the structure of cellulose

Abstract: The DNA fragment encoding the cellulose binding domain of endoglucanase III (CBDEG III) from Trichoderma reesei was subcloned and expressed in E. coli. The CBDEG III had a high affinity for cellulose. The morphological and structural changes of cellulose after treatment with CBDEG III indicated a 17% decrease in number of hydrogen bonds and a 16.5% decrease in crystalline index. X-ray diffraction and IR spectra analyses indicated that the destabilization and breakage of the hydrogen bonds in crystalline cellulose accounted for the non-hydrolytic disruption of the structure of cellulose.

Mutant trichoderma reesei egiii cellulases, dna encoding such egiii compositions and methods for obtaining same

Abstract: The present invention relates to variant EGIII cellulases that have improved stability and/or performance. The variant cellulases have replacements at sensitive residues to improve stability and/or performance.

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Abstract: Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Abstract: Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l−1 were 20 g dry cell wt l−1, 1.9 FPU ml−1 and 4.8 FPU mg−1 protein respectively, while on 40 g glucose l−1 were 25 g dry cell wt l−1, 4.5 FPU ml−1 and 6.2 FPU mg−1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg−1 protein) than was produced by other T. reesei mutants (3.6 FPU mg−1 protein).

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose‐binding domain

Abstract: The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.