| 1. |
- Enebro, Jonas, 1979-, et al.
(författare)
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A New Approach for Studying Correlations between the Chemical Structure and the Rheological Properties in Carboxymethyl Cellulose
- 2007
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 8:10, s. 3253-3257
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Enebro, Jonas, 1979-, et al.
(författare)
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Investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques
- 2009
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Ingår i: Cellulose. - : Springer Science and Business Media LLC. - 0969-0239 .- 1572-882X. ; 16:2, s. 271-280
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Tidskriftsartikel (refereegranskat)abstract
- The benefits of applying cellulose selective enzymes as analytical tools for chemical structure characterization of cellulose derivatives have been frequently addressed over the years. In a recent study the high selectivity of cellulase Cel45A from Trichoderma reesei (Tr Cel45A) was utilized for relating the chemical structure to the flow properties of carboxymethyl cellulose (CMC). However, in order to take full advantage of the enzymatic hydrolysis the enzyme selectivity on the cellulose substrate must be further investigated. Therefore, the selectivity of Tr Cel45A on CMC was studied by chemical sample preparation of the enzyme products followed by mass spectrometric chemical structure characterization. The results strongly suggest that, in accordance with recent studies, also this highly selective endoglucanase is able to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (AGUs). Furthermore, the results also indicate that substituents on the nearby AGUs will affect the hydrolysis.
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| 3. |
- Enebro, Jonas, 1979-, et al.
(författare)
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Liquid Chromatography combined with Mass Spectrometry for the Investigation of Endoglucanase Selectivity on Carboxymethyl Cellulose
- 2009
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 344:16, s. 2173-2181
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Karlsson, Johan, et al.
(författare)
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Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei
- 2002
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 99:1, s. 63-78
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Tidskriftsartikel (refereegranskat)abstract
- 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 kCat 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 @b-glucan and glucomannan were studied. Cel12A hydrolysed @b-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with @b-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.
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| 5. |
- Karlsson, Johan, et al.
(författare)
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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:24, s. 6498-6507
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Tidskriftsartikel (refereegranskat)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 β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-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.
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| 6. |
- Kotiranta, Pia, et al.
(författare)
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Adsorption and Activity of Trichoderma reesei Cellobiohydrolase I, Endoglucanase II, and the Corresponding Core Proteins on Steam Pretreated Willow
- 1999
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Ingår i: Applied Biochemistry and Biotechnology. - 1559-0291. ; 81:2, s. 81-90
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Tidskriftsartikel (refereegranskat)abstract
- The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBH I. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.
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| 7. |
- Penttilä, Paavo A., et al.
(författare)
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Xylan as limiting factor in enzymatic hydrolysis of nanocellulose
- 2013
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 129, s. 135-141
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Rodriguez, Yohama Puentes, et al.
(författare)
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Susceptibility of pre-treated wood sections of Norway spruce (Picea abies) clones to enzymatic hydrolysis
- 2012
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Ingår i: Canadian Journal of Forest Research. - : NRC Research Press. - 0045-5067 .- 1208-6037. ; 42:1, s. 38-46
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Tidskriftsartikel (refereegranskat)abstract
- The structure of softwoods, which confers resistance to degradation through hydrolysis and decay, currently limits their use for the production of biofuels. However, since wood is very heterogeneous, it is possible that differences in wood properties within and between trees could differentially affect its processability. In this research, heartwood (inner) and sapwood (outer) from Norway spruce (Picea abies (L.) Karst.) clones were enzymatically hydrolyzed by Trichoderma viride cellulases after concentrated acid pretreatment. Wood sections with two particle sizes were compared based on their susceptibility to enzymatic hydrolysis, evaluated by assaying the formation of hydrolysis products and measured as reducing sugar yield (RSY). We also studied the relationship between RSY and the susceptibility to Heterobasidion parviporum wood decay and whether these traits are reflected in wood density and yield. Wood from the outer section produced more RSY with higher glucan but lower lignin content than wood from the inner section. Furthermore, susceptibility to enzymatic hydrolysis was positively correlated with H. parviporum wood decay, while both processes were negatively correlated with wood density. Our results revealed the importance of clonal trials for identifying suitable lignocellulosic biomass when considering wood properties and indicate that potential genotypes for the production of biofuels are not necessarily the most productive.
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| 9. |
- Öhgren, Karin, et al.
(författare)
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High temperature enzymatic prehydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production
- 2007
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 40:4, s. 607-613
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Tidskriftsartikel (refereegranskat)abstract
- As a consequence of heightened concern for the greenhouse effect, depleting oil reserves and skyrocketing oil prices, renewable fuels, Such as bioethanol, are becoming increasingly important. Cellulosic biomass like wood, agricultural residue and herbaceous material is a huge, cheap natural resource that can be used for ethanol production. Decreasing production costs through technological development is, however, still needed to make ethanol from cellulosics economically feasible. In this study, the effect on overall ethanol yield of an enzymatic prehydrolysis prior to simultaneous saccharification and fermentation (SSF) of steam pretreated corn stover was investigated. Two enzyme mixtures were utilised. A mixture of commercially available enzymes (Novozymes A/S, Baegersvaerd, Denmark) was compared with a developmental thermo-active cellulase complex produced by Roal Oy (Rajam&i, Finland). The thermoactive preparation comprised three essential cellulases and one xylanase enzyme. The prehydrolysis was, under the conditions evaluated, found to have no or negative effect on the overall ethanol yield. Longer prehydrolysis time resulted in a larger decrease in overall ethanol yield than shorter prehydrolysis. Using the experimental thermo-active enzyme mixture was shown to give a higher glucose concentration after prehydrolysis than when the commercial enzyme mixture was used in similar experiments. The highest ethanol concentration, 33.8 g/L, was reached in the SSF with 11.5% water-insoluble substances using the developmental thermoactive cellulase complex, and 1.8 g/L compressed baker's yeast. This concentration corresponded to 80.2% overall ethanol yield based on the glucose content in the raw material. However, if the xylose present in the beer at the end of the SSF could be fermented to ethanol, another 12.6 g ethanol/L could theoretically be produced (0.51 g ethanol/g xylose). (c) 2006 Elsevier Inc. All rights reserved.
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