Browsing by Author "Sulaeva, Irina"

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Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section
(2024-05-13) Sulaeva, Irina; Sto̷pamo, Fredrik Gjerstad; Melikhov, Ivan; Budischowsky, David; Rahikainen, Jenni L.; Borisova, Anna; Marjamaa, Kaisa; Kruus, Kristiina; Eijsink, Vincent G.H.; Várnai, Anikó; Potthast, Antje
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers. The method can reveal potential differences in fiber properties along the cross-sectional profile of natural or man-made cellulose fibers. In this analytical approach, carbonyl groups are labeled with a carbonyl selective fluorescence label (CCOA), after which thin fiber layers are sequentially dissolved with the solvent system DMAc/LiCl (9% w/v) and analyzed with size exclusion chromatography coupled with light scattering and fluorescence detection. The analysis of these fractions allowed for the recording of the changes in the chemical structure across the layers, resulting in a detailed cross-sectional profile of the different functionalities and molecular weight distributions. The method was optimized and tested in practice with LPMO (lytic polysaccharide monooxygenase)-treated cotton fibers, where it revealed the depth of fiber modification by the enzyme.
The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers
(2024-08-24) Støpamo, Fredrik G.; Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Kruus, Kristiina; Potthast, Antje; Eijsink, Vincent G.H.; Várnai, Anikó
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. Results: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. Conclusions: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.
Indirect determination of partial depolymerization reactions in dialdehyde celluloses (DAC) by gel permeation chromatography of their oxime derivatives
(2023-09) Fliri, Lukas; Simon, Jonas; Sulaeva, Irina; Rosenau, Thomas; Potthast, Antje; Hummel, Michael
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Owing to a supposed quantitative transformation, oximation of dialdehyde cellulose (DAC) with hydroxylamine hydrochloride is commonly employed in chemical DAC analysis, e.g., for the determination of the degree of oxidation (DO) by titration or elemental analysis. In this study, this modification was utilized for the indirect determination of molecular weight distributions (MWD) by gel permeation chromatography (GPC). The presumably quantitative conversion of aldehyde groups in DAC to the corresponding oxime also breaks up the intermolecular and intramolecular hemiacetal crosslinks, which were associated with solubility issues in the DMAc/LiCl solvent system in previous studies. The limits of the procedure and the material's stability during oximation were investigated. For samples with a DO up to approximately 9% a good applicability was observed, before at higher DO values residual crosslinks led to solubility problems. The oximation/GPC protocol was used to examine the development of the MWD in the early stages of DAC formation under different reaction conditions. The time-dependent partial depolymerization of the polymer backbone was observed. Furthermore, the stability of DAC towards different pH conditions ranging from strongly acidic to strongly alkaline was tested. The depolymerization of DAC in alkaline media occurred with concomitant degradation of aldehyde moieties. In turn, DAC proved to be remarkably stable in acidic and neutral solutions up to a pH of 7.
LPMO-Catalyzed Oxidation of Cellulosic Fibers with Controlled Addition of a Reductant and H2O2
(2025-01-13) Marjamaa, Kaisa; Rahikainen, Jenni; Støpamo, Fredrik G.; Sulaeva, Irina; Hosia, Waltteri; Maiorova, Natalia; King, Alistair W.T.; Potthast, Antje; Kruus, Kristiina; Eijsink, Vincent G.H.; Várnai, Anikó
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Cellulose-derived biomaterials offer a sustainable and versatile platform for various applications. Enzymatic engineering of these fibers, particularly using lytic polysaccharide monooxygenases (LPMOs), shows promise due to the ability to introduce functional groups onto cellulose surfaces, potentially enabling further functionalization. However, harnessing LPMOs for fiber engineering remains challenging, partly because controlling the enzymatic reaction is difficult and partly because limited information is available about how LPMOs modify the fibers. In this study, we explored controlling LPMO-mediated fiber oxidation by sequentially adding a reductant (gallic acid, GA) and H2O2, using three different carbohydrate-binding module (CBM)-containing LPMOs. An in-depth analysis of the soluble products and the Mn, Mw, and carbonyl content in the fiber fraction indicates that fiber oxidation can indeed be controlled by adjusting the amount of GA and H2O2 added to the reaction. In particular, at lower overall dosages of GA and H2O2, corresponding to low oxidation levels, fiber oxidation occurs rapidly with almost no release of soluble oxidized products. Conversely, at higher dosages, fiber oxidation levels off, while oxidized oligosaccharides continue to be released and the fibers are eroded. Importantly, next to demonstrating controlled fiber oxidation, this study shows that different cellulose-active LPMOs modify the fibers in different manners.
A mechanistic study on the alleged cellulose cross-linking system: Maleic acid/sodium hypophosphite
(2024-12-15) Lehrhofer, Anna F.; Fliri, Lukas; Bacher, Markus; Budischowsky, David; Sulaeva, Irina; Hummel, Michael; Rosenau, Thomas; Hettegger, Hubert
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
A combination of maleic acid and sodium hypophosphite as a durable press finishing agent has been reported as a safer but equally effective alternative to conventional formaldehyde-based cross-linking agents for applications in cellulose-based fiber and textile finishing. However, the mechanistic details of this system have not yet been fully elucidated to allow optimization of the conditions. Effective cross-linking treatment requires high curing temperatures of ≥160 °C, which enhances oxidative and thermal degradation of cellulose. In this work, the sequential steps of the cross-linking mechanism were investigated both with model compounds and cellulosic substrates. Extensive NMR studies on model compounds revealed several side reactions alongside the synthesis of the targeted cross-linkable moiety. As an alternative, to circumvent side reactions, a two-step procedure was used by synthesizing the cross-linker sodium 2-[(1,2-dicarboxyethyl)phosphinate]succinic acid in a well-defined pre-condensation reaction before application onto the cellulosic substrate. Further, the effect of the cross-linking treatment on the molecular weight distribution of cellulose was studied by gel permeation chromatography, which showed degradation due to maleic acid/sodium hypophosphite treatment. By using sodium 2-[(1,2-dicarboxyethyl)phosphinate]succinic acid and sodium hypophosphite, this degradation could be significantly limited.
New Opportunities in the Valorization of Technical Lignins
(2021-02-18) Balakshin, Mikhail Yu; Capanema, Ewellyn A.; Sulaeva, Irina; Schlee, Philipp; Huang, Zeen; Feng, Martin; Borghei, Maryam; Rojas, Orlando J.; Potthast, Antje; Rosenau, Thomas
A2 Katsausartikkeli tieteellisessä aikakauslehdessä
Sugar-based biorefineries have faced significant economic challenges. Biorefinery lignins are often classified as low-value products (fuel or low-cost chemical feedstock) mainly due to low lignin purities in the crude material. However, recent research has shown that biorefinery lignins have a great chance of being successfully used as high-value products, which in turn should result in an economy renaissance of the whole biorefinery idea. This critical review summarizes recent developments from our groups, along with the state-of-the-art in the valorization of technical lignins, with the focus on biorefinery lignins. A beneficial synergistic effect of lignin and cellulose mixtures used in different applications (wood adhesives, carbon fiber and nanofibers, thermoplastics) has been demonstrated. This phenomenon causes crude biorefinery lignins, which contain a significant amount of residual crystalline cellulose, to perform superior to high-purity lignins in certain applications. Where previously specific applications required high-purity and/or functionalized lignins with narrow molecular weight distributions, simple green processes for upgrading crude biorefinery lignin are suggested here as an alternative. These approaches can be easily combined with lignin micro-/nanoparticles (LMNP) production. The processes should also be cost-efficient compared to traditional lignin modifications. Biorefinery processes allow much greater flexibility in optimizing the lignin characteristics desirable for specific applications than traditional pulping processes. Such lignin engineering, at the same time, requires an efficient strategy capable of handling large datasets to find correlations between process variables, lignin structures and properties and finally their performance in different applications.
A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres
(2024-03-15) Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Støpamo, Fredrik Gjerstad; Khaliliyan, Hajar; Melikhov, Ivan; Rosenau, Thomas; Kruus, Kristiina; Várnai, Anikó; Eijsink, Vincent G.H.; Potthast, Antje
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications. An obstacle for wide implementation of LPMOs in tailored cellulose chemistry is the lack of suitable techniques to precisely monitor the LPMO impact on the polymer. Soluble oxidized cello-oligomers can be quantified using chromatographic and mass-spectrometric techniques. A considerable portion of the oxidized sites, however, remain on the insoluble cellulose fibres, and their quantification is difficult. Here, we describe a method for the simultaneous quantification of oxidized sites on cellulose fibres and changes in their molar mass distribution after treatment with LPMOs. The method is based on quantitative, heterogeneous, carbonyl-selective labelling with a fluorescent label (CCOA) followed by cellulose dissolution and size-exclusion chromatography (SEC). Application of the method to reactions of seven different LPMOs with pure cellulose fibres revealed pronounced functional differences between the enzymes, showing that this CCOA/SEC/MALS method is a promising tool to better understand the catalytic action of LPMOs.
Oxidation of cellulose fibers using LPMOs with varying allomorphic substrate preferences, oxidative regioselectivities, and domain structures
(2024-04-15) Støpamo, Fredrik G.; Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Potthast, Antje; Kruus, Kristiina; Eijsink, Vincent G.H.; Várnai, Anikó
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Lytic polysaccharide monooxygenases (LPMOs) are excellent candidates for enzymatic functionalization of natural polysaccharides, such as cellulose or chitin, and are gaining relevance in the search for renewable biomaterials. Here, we assessed the cellulose fiber modification potential and catalytic performance of eleven cellulose-active fungal AA9-type LPMOs, including C1-, C4-, and C1/C4-oxidizing LPMOs with and without CBM1 carbohydrate-binding modules, on cellulosic substrates with different degrees of crystallinity and polymer chain arrangement, namely, Cellulose I, Cellulose II, and amorphous cellulose. The potential of LPMOs for cellulose fiber modification varied among the LPMOs and depended primarily on operational stability and substrate binding, and, to some extent, also on regioselectivity and domain structure. While all tested LPMOs were active on natural Cellulose I-type fibers, activity on the Cellulose II allomorph was almost exclusively detected for LPMOs containing a CBM1 and LPMOs with activity on soluble hemicelluloses and cello-oligosaccharides, for example NcAA9C from Neurospora crassa. The single-domain variant of NcAA9C oxidized the cellulose fibers to a higher extent than its CBM-containing natural variant and released less soluble products, indicating a more dispersed oxidation pattern without a CBM. Our findings reveal great functional variation among cellulose-active LPMOs, laying the groundwork for further LPMO-based cellulose engineering.
Recent achievements in the valorization of technical lignins
(2018) Balakshin, Mikhail; Capanema, Ewellyn A.; Huang, Zeen; Sulaeva, Irina; Rojas, Orlando; Feng, Martin; Rosenau, Thomas; Potthast, Antje
A4 Artikkeli konferenssijulkaisussa
Spruce milled wood lignin : Linear, branched or cross-linked?
(2020-07-07) Balakshin, Mikhail; Capanema, Ewellyn Augsten; Zhu, Xuhai; Sulaeva, Irina; Potthast, Antje; Rosenau, Thomas; Rojas, Orlando J.
A2 Katsausartikkeli tieteellisessä aikakauslehdessä
The subject of lignin structure, critical for fundamental and practical reasons, is addressed in this study that includes a review of the methods applied to elucidate macromolecular branching. The recently available approaches for determination of the absolute molecular mass of spruce milled wood lignin (MWL) along with the quantification of terminal groups clearly indicate that MWL is significantly branched and cross-linked (with ∼36% lignin units partaking in these linkages). Results from independent methods imply that about half of the branching and crosslinking linkages involve aromatic rings, predominantly 5-5′ etherified units; meanwhile, a significant number of linkages are located in the side chains. Quantitative 13C NMR analyses suggest that the branches involve different aliphatic ether (alkyl-O-alkyl) types at the α- and γ-positions of the side chain, with intact β-O-4 linkages. While the exact structures of these moieties require further investigation, our results point to the fact that conventional lignification theory disagrees with the presence of such key moieties in softwood MWL and the observed high degree of branching/crosslinking. Potential reasons for the noted discrepancies are discussed. This journal is
Surface-Vinylated Cellulose Nanocrystals as Cross-Linkers for Hydrogel Composites
(2025-04-14) Kröger, Marcel; Pääkkönen, Timo; Fliri, Lukas; Lehrhofer, Anna F.; Sulaeva, Irina; Potthast, Antje; Kontturi, Eero
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Cellulose nanocrystal (CNC) fillers have been shown to significantly improve the performance of polymer composites and hydrogels, elevating both strength and toughness. Polymer grafting from the surface of the nanocrystals has been employed to enhance matrix-filler interactions and keep the fillers dispersed within the matrix. However, such approaches often rely on multistep syntheses and diligent process control. Here, we propose modifying the nanocrystal surface to carry vinyl moieties, turning the particles into cross-linking comonomers. Using allyl glycidyl ether in an aqueous modification route, we were able to decorate the CNCs with varying amounts of vinyl moieties. Subsequent dispersion in 2-hydroxy methacrylate and thermally initiated free radical polymerization yielded composite materials that showed superior mechanical performance compared to those obtained from monomeric cross-linkers and unmodified CNCs. The large discrepancies in the observed glass transition temperatures of the obtained materials suggest, however, that the impact of the fillers on the polymerization kinetics is significant and less easily explained.