| 1. |
- Anasontzis, George E, 1980, et al.
(författare)
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Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism.Results: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source.Conclusions: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics. © 2014 Anasontzis et al.; licensee BioMed Central Ltd.
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| 2. |
- Anasontzis, George E, 1980
(författare)
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Biomass-modifying Enzyme Discovery
- 2012
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Ingår i: Oral presentation at the WWSC annual conference, 21-23 November 2012, Uddevalla, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Anasontzis, George E, 1980
(författare)
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Biomass modifying enzymes: From discovery to application
- 2012
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Ingår i: Oral presentation at the Chalmers Life Science AoA conference.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- It has now been realized that the road towards the bio-based economy is a one-way street, leaving gradually the oil-based technology and driving slowly towards a more sustainable society. The current non-biodegradable hydrocarbon fuels and plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic material to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in their subsequent modification, prior to further product development.Our group interests focus on all levels of applied enzyme research of biomass acting enzymes: Discovery, assay development, production and application. Relevant examples will be provided: What is our strategy for discovering novel microorganisms and enzymes from the tropical forests and grasslands of Vietnam? How do we design novel real-world assays for enzyme activity determination? Which are the bottlenecks in the enzymatic cellulose hydrolysis? How enzymes can be used to produce high added value compounds from biomass?
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| 4. |
- Anasontzis, George E, 1980, et al.
(författare)
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Challenges in ethanol production with fusarium oxysporum through consolidated bioprocessing
- 2014
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Ingår i: Bioengineered Bugs. - : Informa UK Limited. - 1949-1018 .- 1949-1026 .- 2165-5979 .- 2165-5987. ; 5:6, s. 393-395
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Tidskriftsartikel (refereegranskat)abstract
- Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.
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| 5. |
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| 6. |
- Anasontzis, George E, 1980, et al.
(författare)
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Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
- 2014
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 30:3, s. 728-735
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Tidskriftsartikel (refereegranskat)abstract
- Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. (c) 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728-735, 2014
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| 7. |
- Anasontzis, George E, 1980, et al.
(författare)
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Enzyme Discovery Platform
- 2012
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Ingår i: WWSC annual conference, 21-23 November 2012, Uddevalla, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Biomass treatment for the separation of its components and the modification of their properties requires a wide range of enzymes. Efficiency matters too, so multiple enzymes of the same activity need to be tested before they can be applied in a larger scale. We have launched the implementation of a strategic plan for the discovery of novel enzymes, starting from the isolation of new fungal strains from Vietnam and leading to the cloning and characterization of the enzymes of interest.
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| 8. |
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| 9. |
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
- Anasontzis, George E, 1980, et al.
(författare)
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Investigating the Yet-Unknown Biomass Degrading and Modifying Enzymes of Aspergillus oryzae
- 2012
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Ingår i: Science and Technology Day, 27 March 2012, Göteborg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The association of plant cell wall components with A. oryzae’s extracellular enzyme machinery was investigated by analyzing the transcriptome profile in relation to the chemical structure of the carbon sources. Strain RIB40 of A. oryzae was cultured on various carbon sources, namely cellohexaose, mannohexaose, xylopentaose, arabinoheptaose, glucohexaose, glucosyl maltotriosyl maltotriose, galactosyl mannotriose, turanose and sophorose, and the transcribed genes were determined with DNA microarrays. The statistically significant genes were selected and potential novel hydrolases were identified.Presently, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion.
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| 15. |
- Anasontzis, George E, 1980, et al.
(författare)
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Investigating the yet-unknown biomass degrading and modifying enzymes of Aspergillus oryzae
- 2011
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Ingår i: WWSC Workshop, 29 November - 1 December, Södertuna, Gnesta, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The associations of plant cell wall components with A. oryzae’s extracellular enzyme machinery were investigated from a chemical perspective using an integrated analysis of the transcriptome profile. We were able to identify novel hydrolases, which we heterologous expressed for subsequent characterization. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion. In this work, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process.
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| 16. |
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| 17. |
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| 18. |
- Anasontzis, George E, 1980, et al.
(författare)
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Rice straw hydrolysis using secretomes from novel fungal isolates from Vietnam
- 2017
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Ingår i: Biomass and Bioenergy. - : Elsevier BV. - 1873-2909 .- 0961-9534. ; 99, s. 11-20
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Tidskriftsartikel (refereegranskat)abstract
- With a production of 39 million metric tons each year, rice is one of the main agricultural products of Vietnam. Thus, rice straw is a significant by-product, whose use in a biorefinery process would contribute to the bio-based transformation of the Vietnamese and South East Asian economy. In order to find novel efficient enzyme mixtures for the hydrolysis of rice straw and other agricultural residues, we took advantage of the rapidly evolving biodiversity of Vietnam and screened 1100 new fungal isolates from soil and decaying plant tissues for their CMCase activity. We selected 36 strains and evaluated them for their cellulases, xylanases, and accessory enzymes activities. Most of these isolates belonged to the genera Aspergillus and Trichoderma. We identified a few promising isolates, such as A. brunneoviolaceus FEC 156, A. niger FEC 130 and FEC 705, and A. tubingensis FEC 98, FEC 110 and FEC 644, whose produced enzyme mixtures released a mass fraction of the sugar content of alkali-treated rice straw higher than 20%, compared to 10% for Trichoderma reesei RUT C-30. We verified that the black Aspergilli are particularly efficient in their saccharification ability. We also identified strains that although they produced low amounts of cellulases and xylanases, their enzyme mixtures had high saccharification efficiencies, indicating the importance of the synergy effect, rather than the amount of enzymes available. Our results highlight the intra-species variation, especially in the Trichoderma genus, regarding the biomass degradation characteristics and the associated range of enzymatic activities.
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| 19. |
- Anasontzis, George E, 1980, et al.
(författare)
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Screening Natural Resources for Enzymes With Wood Degrading and Wood Modifying Properties
- 2011
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Ingår i: Italic6/COST conference, 5–8 September 2011, Viterbo, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The production of high added value compounds from forest and agricultural biomass has become one of the main targets of contemporary carbohydrates research. The renewability of the biomass, the potential use of waste residues and the complete or partial biodegradability of the products have made the whole approach an attractive perspective towards the sustainable and green ideal. However, most of the already developed biomass separation and modification processes are based on chemical reactions at extreme conditions that are costly and often harmful for the environment. Enzymatic and microbial catalyzed processes present an interesting alternative. The development and discovery of novel biological approaches in the modification, degradation and separation of wood biomass is one of the main activities of the Industrial Biotechnology Group at Chalmers University of Technology, also as part of the Wallenberg Wood Science Center (WWSC).Presently, we pursue this aim through a triple approach: •Μultiple enzymatic screening of phytopathogenic and wood degrading filamentous fungi, such as Trametes hirsuta and Penicillium pinophilum, as well as screening newly isolated microorganisms. We seek enzymes with industrially interesting activities and unique properties, such as reactivity under extreme conditions.•Microorganisms efficient in degrading lignocellulose produce enzyme in response to the environmental conditions. In collaboration with Associate Professor Gianni Panagiotou, Center for Biological Sequence Analysis, DTU, we are looking for sequenced, but still unclassified proteins, which are related to the degradation of plant biomass using information from transcriptomics analysis of Aspergillus oryzae grown on different carbon sources.•Novel enzymes can only be identified by new methods. We investigate the properties of synthetic model compounds that can simulate the natural substrates and the implementation of different analytical methods for the identification of the sometimes complex and singular enzymatic activities. In collaboration with Associate Professor Paul Christakopoulos, BIOtechMASS Unit, School of Chemical Engineering, National Technical University of Athens, we also attempt to isolate model compounds from plant cell wall material.
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| 20. |
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| 21. |
- Anasontzis, George E, 1980, et al.
(författare)
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Screening the tropical fungal biodiversity of Vietnam for biomass modifying enzymes, with secretome and transcriptome analyses
- 2013
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Ingår i: 27th Fungal Genetics Conference.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In the bio-based economy concept, the current hydrocarbon fuels and non-biodegradable plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic materials to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in subsequent modifications, prior to further product development. The type of application usually defines the conditions where the reactions should take place. Thus, novel enzymes with variable combined properties, such as different thermotolerance, pH range of activity, substrate specificity and solvent tolerance, still need to be discovered and developed to achieve the highest possible efficiency in each occasion. We took advantage of the rapidly evolving and high biodiversity of the tropics and have been screening various isolates for their cellulases and hemicellulases activities. Promising strains were then cultivated in bioreactors with different carbon sources, such as wheat bran, spruce and avicel and their biomass degrading capacity was analysed through cross species protein identification of their secretome with TMT. Information on the genes involved in the different stages of the fermentation and the carbon source will be acquired with next generation sequencing of the total transcriptome. Interesting transcripts will then be used to heterologously clone and express the respective genes and identify their role in the degradation process.
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| 22. |
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| 23. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Enzyme production and immobilization in mesoporous materials
- 2014
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes display high reactivity and selectivity under normal conditions, but may suffer from denaturation in industrial applications. A strategy to solve this limitation is to immobilize enzymes [1]. Mesoporous silica materials (MPS) have become a common choice as support to immobilized enzymes. MPS offer unique properties such as high enzyme loading and tunable pore size [2].Feruloyl esterase (FAE) is a subclass (EC. 3.1.1.73) of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids [3]. They are also examples of FAEs used for esterification and transesterification [4].From the genomes of Aspergillus glacus and Aspergillus oryzae, some putative FAE were identified. Among them, five were selected for further investigation in order to find a suitable enzyme for catalyzing the reaction of interest. The selected genes were quite distant in an evolutionary tree.The five putative FAEs were cloned into Pichia pastoris and produced by fed-batch fermentation. They were then purified either by IMAC columns or by ion-exchange chromatography. Their activity was assessed against a range of substrate to screen for FAE, tannase and other esterase activities. When the type of the respective enzyme activity was determined, some of them were further characterized. Five new enzymes were recombinantly produced and purified. Their activity type was determined and some of them were immobilized.Enzymes produced in sufficient quantities and having a good free activity were further investigated by immobilization. The selected support for immobilization was mesoporous silica particles (MPS). The conditions of immobilization were investigated and the activity once immobilized was tested and compared to the free one to gain insights on what happens during the immobilization of enzymes. Results were compared to those obtained with a commercially available FAE (E-FAERU, Megazyme).References.[1] Hudson S.; Cooney J.; Magner E., Angew. Chem. Int. Ed. 2008, 47, 8582-8594. [2] Carlsson N.; Gustafsson H.; Thörn C.; Olsson L.; Holmberg K.; Åkerman B. Advances in Colloid and Interface Science 2014, 204, 339-360.[3] Topakas E.; Vafiadi C.; Christakopoulos P. Process Biochemistry 2007, 42, 497-509.[4] Thörn C.; Gustafsson H.; Olsson L. Journal of molecular Catalysis B: Enzymatic 2011, 72, 57-64
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| 24. |
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| 25. |
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| 26. |
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| 27. |
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| 28. |
- McKee, Lauren S., et al.
(författare)
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A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan
- 2016
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant alpha-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me) GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an alpha-l-arabinofuranosidase (AbfA), and a beta-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 alpha-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.
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| 29. |
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| 30. |
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| 31. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Challenges in cellulolytic enzyme production by filamentous fungus Trichoderma reesei on cellulosic materials
- 2012
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Ingår i: Abstract book of Life Science Engineering Area of Advance Conference, Chalmers University of Technology, Gothenburg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Cellulose is the most abundant renewable and biodegradable material on earth, therefore researchers have a great interest in development of cellulose based sustainable energy industry and production of value-added products. Enzymatic hydrolysis of cellulose by cellulolytic enzymes is an essential step in the biomass-to-biofuels and biochemicals process. Some filamentous fungi use cellulose as a nutrient source for their growth and in turn they produce cellulolytic enzymes. The industrial cellulase production is dominated by the filamentous fungus Trichoderma reesei [1]. The use of cellulosic substrates instead of lactose and other expensive substrates, currently employed in industry for cellulolytic enzyme production, would significantly reduce the cost of the process. However, enzyme productivities are significantly lower on cellulose than on lactose. We studied the enzyme production by T. reesei Rut C-30 on the model and industrial cellulosic substrates. Industrial substrates have a high content of cellulose but also contain residual hemicellulose, lignin and some inhibitors. The results of our study will bring insight into bottlenecks of enzyme production on cellulose.During the enzyme production study, we grew T. reesei strain Rut C-30 in submerged fermentations on commercial cellulose Avicel PH101 and industrial-like cellulosic substrates from spruce. These substrates were produced during the process of sodium hydroxide cooking, used in pulp and paper industry. The chemical and physical properties of the substrates were characterized by high performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), gravimetric analysis of lignin and solid-state nuclear magnetic resonance (NMR) technique. We measured protein yields, cellulolytic enzyme activities and examined protein profile. Also, we analyzed the influence of inhibitors on fungal growth and how different substrates affect fungal morphology.Inhibitors, soluble sugars and lignin degradation products did not have any impact on the growth ability of T. reesei Rut C-30 but it did influence the fungal morphology. Industrial-like substrates yielded less enzymes and enzyme adsorption may be one important factor influencing protein yields.
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| 32. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Challenges in cellulolytic enzyme production by filamentous fungus Trichoderma reesei Rut C-30 on cellulosic materials
- 2013
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Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals abstract book.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The industrial cellulase production is dominated by the filamentous fungus Trichoderma reesei. The switch of the carbon sources from lactose, traditionally used for industrial cellulase production, to lignocellulose, such as residual plant materials, could potentially reduce the cost of the enzyme production process. However, enzyme productivities are significantly lower on cellulose than on lactose. We studied the enzyme production by T. reesei strain Rut C-30 on model and industrial cellulosic substrates. Our aim was to understand how different raw materials influence the levels and profiles of the proteins produced. Enzyme production by T. reesei Rut C-30 was studied in submerged cultivations on commercial cellulose Avicel and industrial-like cellulosic substrates from softwood, which mainly consist of cellulose, but also contain residual hemicellulose, lignin and some inhibitors. These substrates were produced by alkaline pulping, used in pulp and paper industry. In order to evaluate hydrolysis and consumption of the substrates by fungal enzymes, the substrates were characterized by HPAEC-PAD and solid-state NMR. Lignin was analyzed by gravimetric method. Protein profile was examined by isobaric tag for relative and absolute quantification (iTRAQ). Inhibitors, soluble sugars and lignin degradation products did not have impact on the growth ability of T. reesei Rut C-30, but the fungal morphology was severely influenced during the growth on industrial-like substrates. Industrial-like substrates yielded less enzymes and enzyme adsorption may be one important factor influencing protein yields in the cultivations. Fungal growth on different substrates resulted in distinct protein profiles.
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| 33. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Enzymatic Hydrolysis of Different Cellulosic and Lignocellulosic Materials: What are the Limiting Factors?
- 2012
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Ingår i: Science and Technology Day, 27 March 2012, Göteborg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulose is the most abundant renewable material on earth and plays an important role in development of sustainable energy industry and production of value-added products. Hydrolysis of lignocellulosic materials is most often performed by enzymes produced by the filamentous fungus Trichoderma reesei, which is the main industrial source for enzyme production. However, hydrolysis of lignocellulose is usually incomplete, but limitations are not well understood. We studied the enzyme profile produced during the fungal growth on model cellulosic and lignocellulosic substrates and their capacity to hydrolyze cellulosic and lignocellulosic substrates with different chemical and physical properties. The results of our study will bring insight into bottlenecks of enzymatic hydrolysis.
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| 34. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Enzyme production and hydrolysis of lignocellulosic materials
- 2011
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Ingår i: Abstract book of PhD course on Industrial Biotechnology for Lignocellulosic Bioprocesses.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic materials are critical for the development of the sustainable global economy because they are cheap, abundant and represent a renewable carbon source that can be used in industrial fermentation processes for production of valuable products such as biofuels or chemicals. Hydrolysis of lignocellulosic materials is performed by enzymes from microorganisms such as fungi or bacteria. Trichoderma reesei is one of the main industrial source for enzyme production. However, the complex structure of lignocellulose requires a proper pretreatment prior enzymatic hydrolysis because substrate accessibility is low and therefore enzymes cannot work efficiently. The ability of fungi to cope with industrial-like conditions is not well understood. My project addresses the question on how lignocellulosic materials influence the enzyme production and cellular metabolism of the filamentous fungus T. reesei strain Rut C-30.
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| 35. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Enzyme production by Trichoderma reesei Rut C-30 followed by enzymatic hydrolysis of different lignocellulosic materials
- 2012
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Ingår i: Abstract book of 11th European Conference on Fungal Genetics, Marburg Germany.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The filamentous fungus Trichoderma reesei is one of the main sources for cellulose degrading enzymes. We study the enzyme profile produced during the fungal growth on cellulosic and lignocellulosic substrates and their capacity to hydrolyze cellulosic and lignocellulosic substrates with different chemical and physical properties. The results will bring insight into the bottlenecks of enzymatic hydrolysis. During the enzyme production study, we grew T. reesei strain Rut C-30 in submerged fermentations on Avicel PH101, commercial cellulose, and industrial-like lignocellulosic substrates from spruce. These substrates were produced during the process of sodium hydroxide cooking, used in pulp and paper industry. Additionally we altered the chemical and physical properties of those substrates by drying and rewetting, treatment of sodium hydroxide and sodium chlorite in order to decrease or increase the surface area and delignify, respectively. We measured cellulolytic enzyme activity by enzymatic assays. Proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The enzymes produced were subsequently used for enzymatic hydrolysis of lignocellulosic substrates and compared to enzymatic hydrolysis of model cellulosic substrates, namely, Avicel PH101, nanocrystaline cellulose, phosphoric acid-swollen cellulose and cotton, which have defined characteristics. The structural properties of the substrates during the different times of hydrolysis were analyzed by solid-state nuclear magnetic resonance (NMR) technique. Dynamics of the hydrolysis was analyzed by quartz crystal microbalance with dissipation (QCM-D) technique. Hydrolysis products were verified by high performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).
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| 36. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates
- 2014
-
Ingår i: Fungal Genetics and Biology. - : Elsevier BV. - 1087-1845 .- 1096-0937. ; 72, s. 64-72
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Tidskriftsartikel (refereegranskat)abstract
- The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel (R) and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS C-13-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.
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| 37. |
- Salazar Pena, Margarita, 1979, et al.
(författare)
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Recombinant galactose oxidase production in fed batch fermentations with Pichia pastoris
- 2010
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Ingår i: WWSC Second International Conference, 23-25 November 2010, Stockholm, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- This study concentrates on the optimization of the cultivation conditions, in order to achieve high galactose oxidase productivity from a recombinant strain of the methylotrophic yeast Pichia pastoris.
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| 38. |
- Stathopoulou, P.M., et al.
(författare)
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Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: Biosynthesis of the corresponding activities
- 2012
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Ingår i: World Journal of Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1573-0972 .- 0959-3993. ; 28:9, s. 2889-2902
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Tidskriftsartikel (refereegranskat)abstract
- The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, β-glucosidase and β-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the β-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of β-glucosidase. Xylanase and β-xylosidase production was practically unaffected by aeration levels.
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| 40. |
- Stépán, Agnes, 1985, et al.
(författare)
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Enzymatic Transformation of Hemicelluloses to Bioplastics
- 2012
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Ingår i: 243rd ACS National Meeting, 25-29 March 2012, San Diego, CA, USA.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Hemicelluloses being the second most abundant biopolymer on Earth are getting more attention in the recent years for having a great potential as a renewable source for materials, fine chemicals and fuels. The focus of this study is to modify molecular architecture of spruce arabinoglucuronoxylan (AGX) and to gain better understanding of structure-material properties relationships. Chemical acetylation and gradient debranching of arabinoxylan has been carried out and material properties have been evaluated. The recent work is more focused on enzymatic acetylation and acylation. Most of our enzymatic modifications are based on enzymes with a proven record in biocatalytic reactions. In parallel, we target the use of more specific acting enzymes as well. These enzymes are commercially not available yet, so this part includes enzyme cloning and production. The desired product is a thermoplastic biomaterial suitable for packaging or as binder / matrix in a biocomposite.
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| 43. |
- Stépán, Agnes, 1985, et al.
(författare)
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Lipases efficiently stearate and cutinases acetylate the surface of arabinoxylan films
- 2013
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 167:1, s. 16-23
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Tidskriftsartikel (refereegranskat)abstract
- This is the first report on successful enzyme catalyzed surface esterification of hemicellulose films. Enzyme catalyzed surface acetylation with vinyl acetate and stearation with vinyl stearate were studied on rye arabinoxylan (AX) films. Different surface analytical techniques (FT-IR, TOF-SIMS, ESCA, CA) show that lipases from Mucor javanicus, Rhizopus oryzae and Candida rugosa successfully surface stearate AX films and that a cutinase from Fusarium solani pisi surface acetylates these films. The specificities of cutinase and lipases were also compared, and higher activity was observed for lipases utilizing long alkyl chain substrates while higher activity was observed for cutinase utilizing shorter alkyl chain substrates. The contact angle analysis showed films with increased initial hydrophobicity on the surfaces.
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| 44. |
- Thuy, Nguyen Thanh, et al.
(författare)
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Screening Tropical Microorganisms for the Discovery of Enzymes for Biomass Modification and Degradation
- 2012
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Ingår i: Science and Technology Day, 27 March 2012, Göteborg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The quest for novel enzymes, with properties suitable for the modification and hydrolysis of biomass in industrial environments, requires the exploration of previously unexplored biotopes and their microorganisms. The tropics are often associated with high biodiversity, both in the seas and the land. This characteristic makes Viet Nam an ideal place for the isolation of novel microorganisms and the subsequent identification of novel enzymes with interesting characteristics.Twenty five fungal strains have been isolated from different biomass rich environments in Viet Nam and have been cultivated in media containing rice bran or Brewer’s spent grain. Crude extracts were isolated and tested for amylase, xylanase and cellulase activity. The thermotolerance and halotolerance of the respective enzymes were also investigated.Future work includes the investigation of cellulose degrading, hemicellulose debranching, lignin carbohydrate complexes cleaving and lignin degrading enzymes, identification of interesting properties and purification, heterologous expression and subsequent characterization of the respective enzymes.
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