The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase.
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ABSTRACT:
SUBMITTER: Halliwell G
PROVIDER: S-EPMC1185320 | biostudies-other | 1970 Jan
REPOSITORIES: biostudies-other
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Project description:1. Cell-free culture filtrates from Trichoderma koningii were concentrated by precipitation with ammonium sulphate between the limits of 20% and 80% saturation. 2. Removal of a low-molecular-weight carboxymethylcellulase (CM-cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton. 3. Further chromatography on DEAE-Sephadex separated a component (C(1)) from the C(x) (CM-cellulase) and beta-glucosidase activities. Separately these components had little ability to produce soluble sugars from cotton, but when recombined in their original proportions this capacity was almost completely recovered. 4. The C(x) component was further fractionated on SE-Sephadex into a fraction containing only CM-cellulase and a fraction showing CM-cellulase and beta-glucosidase activities: the latter two components could be separated by heat treatment. 5. The C(1) component had no swelling factor (S-factor) activity (Marsh, Merola & Simpson, 1953; Reese & Gilligan, 1954) on its own, but it had a synergistic effect on the S-factor activity associated with the CM-cellulase and beta-glucosidase components.
| S-EPMC1186778 | biostudies-other
Project description:A procedure involving affinity chromatography on cellulose was developed for separating enzymic components of the cellulase complex. Cellobiase, carboxymethylcellulase, component C2 and cellobiohydrolase are adsorbed with increasing tenacity, and released, as highly purified components, as the ionic strength of the eluent is decreased.
| S-EPMC1183846 | biostudies-other
Project description:The morphological concept of Trichoderma koningii is found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Phylogenetic analysis utilizing partial sequences of the translation-elongation factor 1 alpha (tef1), as well as fragments of actin and calmodulin genes, indicate that phenotypic characters typical of T. koningii evolved independently in three well-separated main lineages. Combined molecular and phenotype data lead to the development of a taxonomy with the recognition of twelve taxonomic species and one variety within the three lineages. These lineages include: (1) T. koningii and T. ovalisporum and the new species T. caribbaeum var. caribbaeum, T. caribbaeum var. aequatoriale, T. dorotheae, T. dingleyae, T. intricatum, T. koningiopsis, T. petersenii and T. taiwanense; (2) the new species T. rogersonii and T. austrokoningii, and (3) the new anamorph T. stilbohypoxyli.Trichoderma koningii s. str. is an uncommon species restricted to Europe and eastern North America; T. caribbaeum var. aequatoriale, T. koningiopsis, and T. ovalisporum were isolated as endophytes of trunks of Theobroma species in tropical America, and T. ovalisporum from the woody liana Banisteropsis caapi in Ecuador; T. koningiopsis is common in tropical America but was isolated also from natural substrata in East Africa, Europe and Canada, and from ascospores in eastern North America, and as an endophyte in Theobroma species; T. stilbohypoxyli, originally described as a parasite of Stilbohypoxylon species in Puerto Rico, is found to be more common in the tropics, besides an endophytic isolate from Fagus in U.K. The additional new species are known almost exclusively from their teleomorphs. Isolates of T. ovalisporum and T. koningiopsis may have biological control potential. A morphophenetic key and a set of tools for molecular species identification were developed.
| S-EPMC2104733 | biostudies-literature
Project description:1. A purified cellulolytic component C(1) was isolated free from associated activities of the cellulase complex and shown to act as a beta-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component C(x) of the cellulase system is not required for the action of component C(1) (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a K(m) of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu(2+), Zn(2+), phenylmercuric and Fe(3+) are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.
| S-EPMC1165873 | biostudies-other
Project description:1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.
| S-EPMC1184133 | biostudies-other
Project description:1. The C(1) component that was isolated from a Trichoderma koningii cellulase preparation (Wood, 1968) by chromatography on DEAE-Sephadex with a salt gradient was still associated with a trace of CM-cellulase activity (determined by reducing-sugar and viscometric methods). 2. Further chromatography on DEAE-Sephadex, with a pH gradient instead of a salt gradient, provided a C(1) component that could still produce reducing sugars from a solution of CM-cellulose (to a very limited extent), but which could no longer decrease the viscosity (i.e. under the assay conditions employed). 3. No evidence for the non-identity of C(1) component and the trace of CM-cellulase activity could be found when electrofocusing was done in a stabilized pH gradient covering three pH units (pH3-6) or, alternatively, only 0.5 pH unit (pH3.72-4.25). 4. The two protein peaks that were separated by electrofocusing in carrier ampholytes covering only 0.5 pH unit (isoelectric pH values of 3.80 and 3.95) were shown to be isoenzymes of the C(1) component: they differed in the extent to which they were associated with carbohydrate (9% and 33%). 5. The purified C(1) component had little ability to attack CM-cellulose or highly ordered forms of cellulose, but degraded phosphoric acid-swollen cellulose readily: cellobiose was the principal product of the hydrolysis (97%). 6. Dewaxed cotton fibre was degraded to the extent of 15% when exposed to high concentrations of C(1) component over a prolonged period: cellobiose was again the principal sugar present in the supernatant (96%). 7. Cellotetraose and cellohexaose were hydrolysed almost exclusively to cellobiose. 8. Evidence indicates that the C(1) component is a beta-1,4-glucan cellobiosylhydrolase.
| S-EPMC1174006 | biostudies-other
Project description:NBRP: Genome sequencing of Trichoderma koningii JCM 1883
| PRJDB3615 | ENA
Project description:Background:The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. Results:In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high ?-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize ?-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. Conclusions:In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.
| S-EPMC5688634 | biostudies-literature
Project description:The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.
| S-EPMC1163384 | biostudies-other
Project description:Despite being the most important cellulase producer, the cellulase-regulating carbon source signal transduction processes in Trichoderma reesei are largely unknown. Elucidating these processes is the key for unveiling how external carbon sources regulate cellulase formation, and ultimately for the improvement of cellulase production and biofuel production from lignocellulose.In this work, the role of the mitogen-activated protein kinase (MAPK) signal transduction pathways on cellulase formation was investigated. The deletion of yeast FUS3-like tmk1 in T. reesei leads to improved growth and significantly improved cellulase formation. However, tmk1 deletion has no effect on the transcription of cellulase-coding genes. The involvement of the cell wall integrity maintenance governing yeast Slt2-like Tmk2 in cellulase formation was investigated by overexpressing tmk3 in T. reesei ?tmk2 to restore cell wall integrity. Transcriptional analysis found little changes in cellulase-coding genes between T. reesei parent, ?tmk2, and ?tmk2::OEtmk3 strains. Cell wall integrity decreased in T. reesei ?tmk2 over the parent strain and restored in ?tmk2::OEtmk3. Meanwhile, cellulase formation is increased in T. reesei ?tmk2 and then decreased in T. reesei ?tmk2::OEtmk3.These investigations elucidate the role of Tmk1 and Tmk2 on cellulase formation: they repress cellulase formation, respectively, by repressing growth and maintaining cell wall integrity, while neither MAPK regulates the transcription of cellulase-coding genes. This work, together with the previous investigations, suggests that all MAPKs are involved in cellulase formation, while Tmk3 is the only MAPK involved in signal transduction for the regulation of cellulase expression on the transcriptional level.
| S-EPMC5397809 | biostudies-other