| 1. |
- Alkasrawi, Malek, et al.
(författare)
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The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol
- 2003
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:1, s. 71-78
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Tidskriftsartikel (refereegranskat)abstract
- Simultaneous sacchatification and fermentation (SSF) of steam-pretreated wood constitutes an attractive process configuration for ethanol production from biomass. However, the high enzyme addition in SSF contributes to a high process cost. In this study we explore the effect of the non-ionic surfactant Tween-20 as an additive in SSE Tween-20 addition at 2.5 g/l had several positive effects on SSF: (i) the ethanol yield was increased by 8%; (ii) the amount of enzyme loading could be reduced by 50%, while maintaining a constant yield; (iii) the enzyme activity increased in the liquid fraction at the end of SSF, probably by preventing unproductive binding of the cellulases to lignin, which could facilitate enzyme recovery; (iv) the time required to attain maximum ethanol concentration was reduced. Surfactants as an additive in SSF can significantly lower the operational cost of the process. However, less expensive surfactants must be investigated. (C) 2003 Elsevier Science Inc. All rights reserved.
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| 2. |
- Eriksson, Torny, et al.
(författare)
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A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei
- 2002
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Ingår i: Applied Biochemistry and Biotechnology. - 1559-0291. ; 101:1, s. 41-60
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Tidskriftsartikel (refereegranskat)abstract
- It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.
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| 3. |
- Eriksson, Torny
(författare)
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Degradation of wood polysaccharides by fungal glycoside hydrolases
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The enzymatic degradation of wood polysaccharides such as cellulose and hemicellulose is an important process in nature. In addition, cellulases and hemicellulases can be used in industrial applications. Fuel ethanol can potentially be produced from wood by enzymatic hydrolysis of cellulose followed by yeast fermentation of the formed sugars. In this thesis, fungal glycoside hydrolases, cellulases and hemicellulases were studied with the aim of increasing our knowledge of the mechanisms involved in the enzymatic hydrolysis of cellulose and lignocellulose. The focus was mainly on cellulases from the filamentous fungus Trichoderma reesei. However, lignocellulose also contains hemicellulose and studies of hemicellulases are included In Paper I-IV the mechanisms involved in cellulose degradation were investigated. Features of enzymatic cellulose hydrolysis such as synergism, decreasing hydrolysis rate during hydrolysis and the effect of adding surfactants to the hydrolysis mixture were studied. The observed decrease in hydrolysis rate by T. reesei Cel7A could be explained by unproductive binding of enzymes to the substrate. Furthermore, an extension of the existing model explaining end-exo synergism is presented and discussed. The use of surfactant to increase efficiency of the enzymatic cellulose hydrolysis is evaluated and a mechanism explaining the effect of surfactant is discussed. In addition, the major components of the cellulase system from the filamentous fungus Penicillium brasilianum was purified and characterised. This thesis elucidates the need to use complex polysaccharide substrate in order to understand the mechanisms of degradation of natural complex substrates. O-acetyl-galactoglucomannan, the major softwood hemicellulose, isolated from spruce wood was used to study enzyme specificity and other biochemical properties (Paper V). Moreover, the function of the carbohydrate-binding module of the T. reesei b-mannanase Man5A was investigated (Paper VI). It was concluded that the carbohydrate-binding module of Man5A bound to cellulose but had an important function in potentiate the mannan hydrolysis of substrates containing both mannan and cellulose. The majority of fungal glycoside hydrolases are glycoproteins. In Paper VII the N- glycosylation of T. reesei Cel7B was investigated using mass-spectrometry. N-glycosylation sites on the catalytic module were identified and it was shown that large parts of the observed heterogeneity of the enzyme preparation was caused by heterogeneity in N-glycan structures.
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| 4. |
- Eriksson, Torny, et al.
(författare)
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Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:7, s. 1266-1276
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
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| 5. |
- Eriksson, Torny, et al.
(författare)
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Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose
- 2002
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 31:3, s. 353-364
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.
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| 6. |
- Hägglund, Per, et al.
(författare)
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A cellulose-binding module of the Trichoderma reesei @b-mannanase Man5A increases the mannan-hydrolysis of complex substrates
- 2003
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 101:1, s. 37-48
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Tidskriftsartikel (refereegranskat)abstract
- Endo-@b-1,4-d-mannanases (@b-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The @b-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5A@DCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5A@DCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.
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| 7. |
- Jørgensen, Henning, et al.
(författare)
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Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888
- 2003
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 32:7, s. 851-861
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l−1 cellulose and 10 g l−1 xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.
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