Showing papers on "Trichoderma reesei published in 2004"
TL;DR: The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.
508 citations
TL;DR: A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae, indicating that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished.
Abstract: A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
362 citations
TL;DR: Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme‐substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzymatic complex is nonproductive.
Abstract: The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.
266 citations
TL;DR: It was found that on the same cellulose basis, the cellulase activity and yield produced were comparable with that on purified cellulose, and the produced cellulase could effectively hydrolyze the corn cob residue.
232 citations
TL;DR: The extent of mycotoxin tests in food enzyme preparations should be judged on a case-by-case basis, through a careful evaluation based on knowledge of taxonomy, biochemistry, and genetics.
194 citations
TL;DR: The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.
Abstract: Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l−1). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l−1. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.
188 citations
TL;DR: The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei.
183 citations
Patent•
28 May 2004TL;DR: In this article, two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and another encoding an acetylxylanesterase are described.
Abstract: Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.
181 citations
TL;DR: It is demonstrated that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.
Abstract: Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.
159 citations
TL;DR: Three specific xyloglucanases isolated from Aspergillus japonicus, Chrysosporium lucknowense and Trichoderma reesei may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanase.
143 citations
TL;DR: Two mediators, N‐hydroxy‐N‐phenylacetamide and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined.
Abstract: The impact of oxidative modification and partial removal of lignin by laccase-mediator treatments on the enzymatic hydrolysis of steam-pretreated softwood (SPS) was evaluated. Two mediators, N-hydroxy-N-phenylacetamide (NHA) and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined. Both simultaneous and sequential combinations of laccase-mediator treatments with commercial cellulases increased the sugar yield in the enzymatic hydrolysis of SPS. The maximal increase was 21% when a sequential treatment was applied. Laccase treatment alone was also shown to improve hydrolysis. NHA oxidized by laccase inhibited significantly the cellulases of Trichoderma reesei, but the presence of the solid substrate protected the activities against oxidative inactivation. Surface analysis of the lignocellulosic substrate before and after the laccase and cellulase treatments revealed an enrichment of lignin and an increase of carboxylic groups on the surface of the hydrolysis residue.
TL;DR: The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions, and mannophosphorylation may be regulated intracellularly by alpha-(1-->2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.
Abstract: The glycosylation of Cel7A (CBH I) from Trichoderma reeseivaries considerably when the fungus is grown under differentconditions. As shown by ESI-MS and PAG-IEF analyses ofboth intact protein and the isolated catalytic core module, themicroheterogeneity originates mainly from the variable ratioof single N-acetylglucosamine over high-mannose structureson the three N-glycosylation sites and from the presence orabsence of phosphate residues. Fully N- and O-glycosylatedCel7A can only be isolated from minimal medium and prob-ablyreflectstheinitialcomplexityoftheproteinonleavingtheglycosynthetic pathway. Extracellular activities are responsi-ble for postsecretorial modifications in other cultivation con-ditions: a-(1!2)-mannosidase, a-(1!3)-glucosidase and anEndo H type activity participate in N-deglycosylation (core),whereas a phosphatase and a mannosidase are probablyresponsible for hydrolysis of O-glycans (linker). The effectsare most prominent in corn steep liquor–enriched media,where the pH is closer to the pH optimum (5–6) of theseextracellular hydrolases. In minimal medium, the low pH andthepresenceofproteasescouldexplainfortheabsenceofsuchactivities. On the other hand, phosphodiester linkages in thecatalytic module are only observed under specific conditions.The extracellular trigger is still unknown, but manno-phosphorylationmayberegulatedintracellularlybya-(1!2)-mannosidases and phosphomannosyl transferases competingfor the same intermediate in the glycosynthetic pathway.Key words: Cel7A/endoglycosidase/N- andO-glycosylation/postsecretorial modifications/Trichoderma reeseiIntroductionCellulases belonging to different glycosyl hydrolase familiesvery often exhibit a multidomain structure (Coutinhoand Henrissat, 1999): a catalytic domain (core) and acarbohydrate-binding module are separated by a linkerpeptide rich in proline, serine, and threonine. Fungalcellulases carry posttranslational modifications on bothdomains: Whereas the linker peptide is highly O-glycosylated, N-glycosylation seems to be restricted to thecore. Filamentous fungi typically synthesize short-chain O-and N-glycans, resembling the mammalian high-mannosetype rather than the hyperglycosylated yeast structures.The glycosylation of cellobiohydrolase I (CBH I, Cel7A),a cellulase abundantly expressed by most Trichodermareesei strains, has been studied particularly well(Table I). Trichoderma cellulases appear in several isoformswith similar catalytic and adsorption properties (Medveet al., 1998), and it has been shown that both N- andO-glycans account for the many isoforms of Cel7A(Pakula et al., 2000).Detailed structural investigations (Harrison et al., 1998;Klarskov et al., 1997) revealed the N-glycosylation inT. reesei Cel7A from respectively strains QM9414 andALKO2877 (derived from QM9414): Single GlcNAc resi-dues in three (Asn45, Asn270, and Asn384) out of fourpotential sites were characterized in the catalytic domain.More complex N-glycan structures were observed withCel7A from the hyperproducing mutant Rut-C30 strain(Maras et al., 1997). The majority are monoglucosylatedhigh-mannose glycans (GlcMan
TL;DR: The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement.
Abstract: The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of ≈ 60 A × 40 A × 50 A and an overall β-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.
TL;DR: Results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well, and agree with the fact that ethanol induces laccase gene expression in some fungi.
Abstract: Filamentous fungi belonging to the homobasidiomycetes offer great potential for industrial and medical applications. They secrete proteins into their culture media with activities or in amounts that are not found in other fungi. For instance, homobasidiomycetes produce various metalloenzymes, such as laccases, which are attractive candidates for a wide variety of applications. These enzymes degrade a large number of recalcitrant pollutants and are a biological and environmentally friendly alternative to the highly contaminating pulping and bleaching treatments of the paper and pulp industries (3, 4). Until now, the expression of basidiomycete metalloenzymes in ascomycete production systems such as Aspergillus ssp. and Trichoderma reesei has had limited success (6). Therefore, basidiomycetes should be developed as hosts for large-scale protein production. The white rot fungus Pycnoporus cinnabarinus is an attractive candidate in this respect. This basidiomycete was selected for its ability to efficiently degrade lignin and to transform lignin-derived compounds such as ferulic acid into vanillin (9, 11, 22). P. cinnabarinus has a simple ligninolytic system. Neither lignin peroxidase nor manganese peroxidase activity has been detected, but laccase is produced (9). Two laccase genes have been cloned, i.e., lcc3-1 or the allelic form lac1 (10, 23) and lcc3-2 (34). Until now, transformation procedures and expression systems for P. cinnabarinus were not available. This was part of the subject of this study.
Classical and molecular genetics have been well established for Schizophyllum commune, which can be considered a model system for the homobasidiomycetes. S. commune was transformed to phleomycin and hygromycin resistance by use of the regulatory sequences of the GPD (glyceraldehyde-3-phosphate dehydrogenase) gene (26, 27). Apart from the GPD promoter, the SC3 promoter can also be used for high-level gene expression (36). The former promoter is constitutively expressed, whereas the monokaryon-specific SC3 promoter is expressed only after a few days of growth. mRNA accumulation in S. commune does not only depend on the promoter used but also depends on the presence of introns in or near the coding sequence of the gene (18, 26). Moreover, AT-rich regions within the coding sequence cause premature termination, resulting in truncated mRNAs (28). Full-length mRNAs have been produced by increasing the GC content in such a region (26).
For this study, a transformation and expression system for P. cinnabarinus was developed. This system was used to produce high levels of the homologous laccase lac1.
TL;DR: The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.
Abstract: Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.
TL;DR: All the fungi investigated showed coinduction of cellulolytic and xylanolytic enzymes during growth on cellulose as well as on xylan, and the highest filter paper activity was measured after cultivation of Penicillium brasilianum IBT 20888 on cellulOSE.
Abstract: For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commenly used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamentous fungi from genus Penicillium and compared with that of T. reesei. Either Solka-Floc cellulose or oat spelt xylan was used as carbon source in shake flask cultivations. All the fungi investigated showed coinduction of cellulolytic and xylanolytic enzymes during growth on cellulose as well as on xylan. The highest filter paper activity was measured after cultivation of Penicillium brasilianum IBT 20888 on cellulose.
TL;DR: It is demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.
TL;DR: Gal1 is a member of the GHMP family and shows conservation of a Gly/Ser rich region involved in ATP binding and amino acids responsible for galactose binding as mentioned in this paper.
Abstract: Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.
TL;DR: Cellulase preparations produced by the cel45A transformants performed well at neutral pH in stone-washing of denim fabric and caused considerably less backstaining as compared to the acid cellulase product of T. reesei.
Patent•
19 Mar 2004
TL;DR: In this article, a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1) have been revealed.
Abstract: Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.
TL;DR: The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III, and the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.
TL;DR: In this paper, sunflower hulls were hydrolyzed with Trichoderma reesei Rut C 30 cellulase and showed 59.8% saccharification, achieving a maximum ethanol yield of 0.454 g/g.
Abstract: Pretreated sunflower hulls hydrolyzed with Trichoderma reesei Rut C 30 cellulase showed 59.8% saccharification. Enzymatic hydrolysate concentrated to 40 g/l reducing sugars was fermented with Saccharomyces cerevisiae var. ellipsoideus under optimum conditions of time (24 h ) , pH (5.0), temperature (30°C) and inoculum size, and it showed a maximum ethanol yield of 0.454 g/g . Ethanol production scaled up in 1 and 15 l fermentors under optimum conditions revealed maximum ethanol yields of 0.449 and 0.446 g/g , respectively.
TL;DR: New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels.
Abstract: Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.
TL;DR: Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction.
TL;DR: The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) is encoded by several cellobiohydrolase, endoglucanase and beta-glucosidase genes, which are co-ordinately expressed upon induction by cellulose or the disaccharide sophorose.
TL;DR: It is demonstrated that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan, and the celF gene, located in the cellulosome-like gene cluster, was cloned in Escherichia coli and antibodies were raised against the overproduced CelF protein.
Abstract: Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of beta-1,3- and beta-1,4-linked beta-D-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.
TL;DR: The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II and the combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.
Abstract: Disulphide bridges were introduced in different combinations into the N-terminal region and the single α-helix of mesophilic Trichoderma reesei xylanase II (TRX II). We used earlier disulphide-bridge data and designed new disulphide bridges for the combination mutants. The most stable mutant contained two disulphide bridges (between positions 2 and 28 and between positions 110 and 154, respectively) and the mutations N11D, N38E, and Q162H. With a half-life of ~56 h at 65°C, the thermostability of this sevenfold mutant was ~5,000 times higher than that of TRX II, and the half-life was 25 min even at 75°C. The thermostability of this mutant was ~30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.
TL;DR: This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp.
Abstract: A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reeseistrains grown in minimalmedia was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were however found in proteins from each strain and these probably occur on both the a1-3 and the a1-6 branch of the highmannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore the predominant N-glycans on Cel7A can be represented as (ManP)0‐1GlcMan7‐8GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man1‐2P)0‐1‐2Man5‐6‐7GlcNAc2 for the wild-type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the a1-3 branch apparently hinders a second mannophoshoryl transfer.
TL;DR: In this paper, a microbial consortium of Trichoderma reesei AS3.3711, Aspergillus niger 3.316 and Saccharomyces cerevisiaes AS2.399 was constructed to decomposed rice chaff on the basis of the characters of each microorganism and the mechanism of cellulases.
TL;DR: Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs, indicating that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolisms in fungi.
Abstract: l-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of l-arabinose, and genetic evidence also shows that it is involved in the catabolism of d-xylose in xylitol dehydrogenase (xdh1) mutants and of d-galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (l-arabinitol) and hexitols (d-allitol, d-sorbitol, l-iditol, l-mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest kcat/Km for l-arabinitol. However, it oxidized galactitol and d-talitol at C4 exclusively, yielding l-xylo-3-hexulose and d-arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d-sorbitol. A lad1 gene disruptant was almost unable to grow on l-arabinose, grew extremely weakly on l-arabinitol, d-talitol and galactitol, showed reduced growth on d-sorbitol and d-galactose and a slightly reduced growth on d-glucose. The weak growth on l-arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of l-arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi.