Search

The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow-loop setup with in-line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off-line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield., (© 2014 American Institute of Chemical Engineers.)

The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes. The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan. When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC-MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, Cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.

Two model sodium carboxymethyl celluloses (CMC) with similar monomer composition but with significant differences in the viscoelastic properties, that could not be assigned to variations in the average molar mass or molar mass distribution, were investigated with respect to the fraction of nonsubstituted cellulose segments in the polymers. The CMCs were hydrolyzed by a purified highly selective endoglucanase. The average molar mass and molar mass distribution of the enzyme products, as measured by size-exclusion chromatography with online multi-angle light scattering and refractive index detection (SEC/MALS/RI), revealed that the enzyme-catalyzed hydrolysis was more effective on one of the CMCs. To investigate whether this was due to a higher fraction of nonsubstituted cellulose segments in the polymer, the concentrations of nonsubstituted enzyme products, e.g., cellotetraose and cellopentaose, were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was concluded that the two CMCs displayed significant differences in the fraction of nonsubstituted cellulose segments. Furthermore, the CMC with the strongest attractive intermolecular interactions, according to rheometry, also contained the highest fraction of nonsubstituted cellulose segments.

Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates. Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The k(Cat) values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases' capacity to hydrolyse beta-glucan and glucomannan were studied. Cel12A hydrolysed beta-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with beta-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase., (Copyright 2002 Elsevier Science B.V.)

Background: Recent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BAL process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates. Results: We show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic CTec3. Conclusions: In this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatic liquefaction was studied by measuring continuously the flowability change of high-solids lignocellulose substrates using a real time viscometric method. Hydrolysis experiments of hydrothermally pretreated wheat straw were carried out with purified enzymes from Trichoderma reesei; Cel7A, Cel6A, Cel7B, Cel5A, Cel12A and Xyn11A. Results obtained at 15% (w/w) solids revealed that endoglucanases, in particular Cel5A, are the key enzymes to rapidly reduce the viscosity of lignocellulose substrate. Cellobiohydrolases had only minor and the xylanase practically no effect on the viscosity. Efficient, fast liquefaction was obtained already at a dosage of 1.5mgofCel5A/gdrysolids. Partial replacement or supplementation of Cel5A by the other major hydrolytic enzymes did not improve the liquefaction. The reduction of viscosity did not correlate with the saccharification obtained in the same reaction, suggesting that efficient liquefaction is rather dependent on the site than the frequency of enzymatic cleavages. [ABSTRACT FROM AUTHOR]

Abstract: The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components. Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments. [Copyright &y& Elsevier]

Investigates the degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) by analyzing the insoluble celulose fragments remaining after enzymatic hydrolysis. Single major peak exhibited by cotton cellulose incubated with EGI; Assessment of solubilization of the cellulose during enzyme attack.

Abstract: Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw. [Copyright &y& Elsevier]

Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these-two in glycoside hydrolase family 5, one in family 10, and one in family 12-have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatic hydrolysis of lignocellulosic substrates is one of the limiting steps in second generation bio-ethanol production. The effects of addition of poly(ethylene glycol) 4000 (PEG) on the efficiency and adsorption of cellulase components during the 72-h hydrolysis of steam-pretreated spruce were investigated. It was found that addition of PEG increased the amount of the enzyme components in the hydrolysis supernatant to varying degrees. As an effect of PEG addition the concentration of free proteins increased by 36% and the free overall cellulolytic activity (FPA) in the liquid fraction by 51%. After 72-h hydrolysis with PEG 48–68% higher activities towards 4-methylumbelliferyl-saccharide substrates were recovered. The results suggest that the effect of PEG is based on the decrease of non-productive enzyme binding. The structure of the hydrolysis residues was studied by 31P NMR. [Copyright &y& Elsevier]