| 1. |
- Aldaeus, Fredrik, et al.
(author)
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Characterization of pulp with high enzymatic hydrolyzability
- 2014
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In: 13th European Workshop on Lignocellulosics and Pulp (EWLP 2014) book of abstracts.
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Conference paper (other academic/artistic)abstract
- Conversion of biomass to biofuels is currently an area that attracts large interest, and lignocellulosic biomass offers the abundance and environmental attributes that can potentially support large-scale biofuel production as an alternative to petroleum-based transportation fuel.In a recent project, Innventia has developed wood based pulps optimized for conversion to biofuels. These novel pulps were produced to target a high level of enzymatic hydrolyzability. To assess the hydrolyzability of these pulps, a laboratory protocol has been established usingan enzyme mixture containing Celluclast 1.5L and Novozyme 188 with an activity of 10 FPU/g pulp (Andersen 2007). Results obtained using this protocol are assumed to be relevant for industrial conditions. In addition to assessment of the produced pulps, the results havebeen compared to commercial cellulose substrates and pulps of a variety of grades.Furthermore, supramolecular properties – specific surface area and average pore size – were determined by an in-house method utilizing solid state nuclear magnetic resonance (Larsson et al. 2013). Kappa numbers, limiting viscosities, ISO-brightness and carbohydrate compositions were determined using standard methods. Molecular mass distributions of cellulose tricarbanilates were determined by size exclusion chromatography with tetrahydrofuran mobile phase (Drechsler et al. 2000).The presentation will discuss the influence of chemical, macromolecular and supramolecular properties of commercial and novel pulp grades on the enzymatic hydrolyzability. Theprotocol used to assess of enzymatic hydrolyzability will be proposed as a benchmark test.
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| 2. |
- Aldaeus, Fredrik, et al.
(author)
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The supramolecular structure of cellulose-rich wood pulps can be a determinative factor for enzymatic hydrolysability
- 2015
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In: Cellulose. - : Springer Science and Business Media LLC. - 0969-0239 .- 1572-882X. ; 22:6, s. 3991-4002
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Journal article (peer-reviewed)abstract
- The enzymatic hydrolysability of three industrial pulps, five lab made pulps, and one microcrystalline cellulose powder was assessed using commercial cellulolytic enzymes. To gain insight into the factors that influence the hydrolysability, a thorough characterization of the samples was done, including their chemical properties (cellulose content, hemicellulose content, lignin content, and kappa number), their macromolecular properties (peak molar mass, number-average molar mass, weight-average molar mass, polydispersity, and limiting viscosity) and their supramolecular properties (fibre saturation point, specific surface area, average pore size, and crystallinity). The hydrolysability was assessed by determination of initial conversion rate and final conversion yield, with conversion yield defined as the amount of glucose in solution per unit of glucose in the substrate. Multivariate data analysis revealed that for the investigated samples the conversion of cellulose to glucose was mainly dependent on the supramolecular properties, such as specific surface area and average pore size. The molar mass distribution, the crystallinity, and the lignin content of the pulps had no significant effect on the hydrolysability of the investigated samples.
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| 3. |
- Brander, Søren, et al.
(author)
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Biochemical evidence of both copper chelation and oxygenase activity at the histidine brace
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.
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| 4. |
- Larsson, Per Tomas, et al.
(author)
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Characterization of cellulose supramolecular structure using solid-state NMR
- 2014
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In: Analysdagarna book of abstracts.
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Conference paper (other academic/artistic)abstract
- Cellulose I isolated from wood in the form of cellulose-rich fibres, i.e. as a pulp, is a widely used raw material that holds a potential for further and more versatile use. Due to its abundance cellulose can be a benign replacement for many materials used in everydaycommodities.Isolated cellulose I is associated with a complex supramolecular structure (in the nanometresdimensional range), and in the case of cellulose-richfibres it is also associated with a complex fibre wall morphology (typical wood fibres are millimetres long and tenths of micrometres wide).The main advantage of using cellulose-rich fibres is an existence of a worldwide industry which has the processing equipment and the know-how necessary for efficient handling and processing of wood-based pulps.Utilization of cellulose I is dependent on the reactivity of the cellulose substrate, here the term reactivity is used in a broad sense. Enzymatic conversion of cellulose-rich fibres to sugars or the dissolution of cellulose for textile fibre manufacture is two examples where different aspects of the cellulosereactivity are important for efficient processing.Several methods for characterizing various aspects of cellulose are available. The degree of polymerization and the degree of cellulose crystallinity are two examples. In the case of cellulose-rich fibres its carbohydrate composition can be of importance. Traditionally lessattention has been paid to the supramolecular characteristics of cellulose although they are in a dimensional range that could exert an influence on the chemistry used.The present work deals with the characterization of the supramolecular properties of cellulose and cellulose-rich fibres and illustrates some examples where the supramolecular structure of the cellulose is a controlling factor for its reactivity. Most of the presented work is based on CP/MAS 13 C-NMR measurements. Using this technique it has been shown that robust measurements of cellulose nanostructures such aslateral fibril dimensions and lateral fibril aggregate dimensions can be obtained and how subsequently the specific surface area of the cellulose in a water-swollen state can be estimated. Moreover, by combining NMR resultswith measurements of the amount of water located inside a water-swollen fibre wall, estimates of the average fibre wall pore size can be obtained. Such results have beenrelated to data from enzymatic hydrolysis of cellulose-richfibres to illustrate the influence of supramolecular structure on enzymatic reactivity.
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| 5. |
- Peciulyte, Ausra, 1986, et al.
(author)
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Cellulolytic enzyme interaction with lignocellulose. Insight to factors limiting enzymatic hydrolysis
- 2013
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In: Gordon Conference: Cellulosomes, Cellulases & Other Carbohydrate Modifying Enzymes internal database.
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Conference paper (other academic/artistic)abstract
- Liberation of fermentable soluble sugars from lignocellulosic biomass during the course of enzymatic hydrolysis is the major obstacle to large-scale implementation of biorefineries due to high cost of enzymes. Enzymatic hydrolysis of lignocellulosic biomass is often incomplete and, therefore, it is of great importance to understand the limitations of the process. Among the limitations of enzymatic hydrolysis, structural properties of lignocellulose have an effect of enzymatic hydrolysis efficiency. Currently, there is a lack of direct methods for visualization and quantification of spatial polymer distribution in lignocellulosic biomass and monitoring of interactions between cellulose degrading enzymes and the substrate. The focus of the work was (i) structural characterization of lignocellulose during the course of hydrolysis and (ii) to provide a more detailed understanding of cellulolytic enzyme interaction with lignocellulose. The overall aim was to understand the limitations in enzymatic hydrolysis of lignocellulosic biomass.Enzymatic hydrolysis was studied on industrial-like lignocellulosic and cellulosic substrates, resulting from alkaline pulping and steam explosion of spruce. Enzymatic hydrolysis of lignocellulosic substrates was compared to enzymatic hydrolysis of model cellulosic substrates. Enzymatic hydrolysis of the substrates was performed with commercial enzyme mixture Celluclast 1.5 L and also with designed enzyme mixtures, consisting of mono-component enzymes. The structural properties of the substrates during an incrementing time of hydrolysis were analyzed by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, Coherent Anti-Strokes Raman Scattering (CARS) and Second Harmonic Generation (SHG) microscopy. Hydrolysis products were verified by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Dynamics of the hydrolysis was analyzed by Quartz Crystal Microbalance with Dissipation (QCM-D) technique.
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| 6. |
- Peciulyte, Ausra, 1986, et al.
(author)
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Challenges in cellulolytic enzyme production by filamentous fungus Trichoderma reesei on cellulosic materials
- 2012
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In: Abstract book of Life Science Engineering Area of Advance Conference, Chalmers University of Technology, Gothenburg, Sweden.
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Conference paper (other academic/artistic)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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| 7. |
- Peciulyte, Ausra, 1986, et al.
(author)
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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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In: 35th Symposium on Biotechnology for Fuels and Chemicals abstract book.
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Conference paper (other academic/artistic)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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| 8. |
- Peciulyte, Ausra, 1986, et al.
(author)
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Critical redox processes during enzymatic saccharification of plant biomass: Lytic polysaccharide monooxygenases at play
- 2017
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In: Cellulases & Other carbohydrate-Active Enzymes, Gordon Research Conference.
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Conference paper (other academic/artistic)abstract
- The recent discovery of redox-active enzymes termed lytic polysaccharide monooxygenases (LPMOs) has led to a significant improvement in the efficiency of enzymatic hydrolysis and thus in the process of the production of biofuels. During saccharification of lignocellulose reactive oxygen species (•OH-; •O2-; H2O2) can be generated as an outcome of redox processes via enzymatic and non-enzymatic routes. These reactive oxygen species are toxic for the enzymes.In this project, redox processes of lignocellulosic biomass during enzymatic saccharification were investigated. A rapid decrease of pH was observed during lignocellulose incubation with and without enzymes which is an indication of redox reactions happening in the complex lignocellulosic material. We do not think that acetate release from hemicellulose alone could explain a decrease in pH values. Carbon dioxide release was observed in lignocellulose during incubation with and without enzymes. There was less carbon dioxide released in the enzyme mixtures where LPMO was present after two days of saccharification. Addition of catalase, which terminates chains of radical chemistry by the dismutation of H2O2 into water and O2, resulted in the improvement of enzymatic saccharification and reduction of acidifying reactions taking place.
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| 9. |
- Peciulyte, Ausra, 1986, et al.
(author)
-
Critical redox processes during enzymatic saccharification of plant biomass: Lytic polysaccharide monooxygenases at play
- 2017
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In: Science & Technology Day electronic book.
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Conference paper (other academic/artistic)abstract
- s a result of the increasing burden on the environment and the scarcity of natural resources, we need to find new ways of supplying a growing population with products for daily life. In a bio-based economy we want to produce bioplastics, biochemicals and biofuels from plant biomass (lignocellulose). One of the reasons why lignocellulose is interesting is that it could serve as an energy source for microorganisms, which could be used to produce many different products of interest. However, the source of energy in lignocellulose is not readily available. We need to use certain enzymes, known collectively as cellulases, which are produced by other microorganisms, such as bacteria and filamentous fungi, to degrade lignocellulose into glucose, which can serve as an energy source.The recent discovery of enzymes termed lytic polysaccharide monooxygenases has led to a significant improvement in the efficiency of enzymatic hydrolysis and thus in the process of the production of biofuels. These enzymes are capable of breaking glycosidic bonds using oxidative mechanism which has not been known until recently.In this project I am focused on addressing how the application of these redox- active enzymes can reach its full potential. More specifically, I am investigating redox processes of lignocellulosic biomass during enzymatic saccharification. I combined electrochemistry (cyclic voltammetry), gas chromatography and high performance liquid chromatography techniques to investigate the saccharification of lignocellulose process.
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| 10. |
- Peciulyte, Ausra, 1986, et al.
(author)
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Enzymatic hydrolysis of cellulose: linking hydrolyzability with cellulose characteristics
- 2014
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In: Science and Technology Day book of abstracts.
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Conference paper (other academic/artistic)abstract
- Liberation of fermentable soluble sugars from cellulosic biomass during the course of enzymatic hydrolysis is the major obstacle to large-scale implementation of biorefineries due to high cost of enzymes. Enzymatic hydrolysis of cellulosic biomass is often incomplete and, therefore, it is of great importance to understand the limitations of the process. Among the limitations of enzymatic hydrolysis, structural properties of cellulose have an effect of enzymatic hydrolysis efficiency. The focus of the present work was structural characterization of cellulose during the course of hydrolysis which indirectly gives information about the interaction between the enzymes and the substrate. The overall aim was to understand the limitations in enzymatic hydrolysis of cellulosic biomass.Enzymatic hydrolysis was studied on industrial-like cellulosic substrates, resulting from alkaline pulping of softwood. Enzymatic hydrolysis of cellulosic substrates was compared to enzymatic hydrolysis of model cellulosic substrates. Enzymatic hydrolysis of the substrates was performed with commercial enzyme mixture Celluclast 1.5 L. The structural properties of the substrates during an incrementing time of hydrolysis were analyzed by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, Coherent Anti-Strokes Raman Scattering (CARS) and Second Harmonic Generation (SHG) microscopy. Hydrolysis products were verified by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).
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