Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery

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Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N–OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)–(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.Peer reviewe

Pesticide Residue Fast Screening Using Thermal Desorption Multi-Scheme Chemical Ionization Mass Spectrometry (TD-MION MS) with Selective Chemical Ionization

In this work, the detection characteristics of a large group of common pesticides were investigated using a multi-scheme chemical ionization inlet (MION) with a thermal desorption unit (Karsa Ltd.) connected to an Orbitrap (Velos Pro, Thermo Fisher Scientific) mass spectrometer. Standard pesticide mixtures, fruit extracts, untreated fruit juice, and whole fruit samples were inspected. The pesticide mixtures contained 1 ng of each individual target. Altogether, 115 pesticides were detected, with a set of different reagents (i.e., dibromomethane, acetonylacetone, and water) in different polarity modes. The measurement methodology presented was developed to minimize the common bottlenecks originating from sample pretreatments and nonetheless was able to retrieve 92% of the most common pesticides regularly analyzed with standardized UHPLC-MSMS (ultra-high-performance liquid chromatography with tandem mass spectrometry) procedures. The fraction of detected targets of two standard pesticide mixtures generally quantified by GC-MSMS (gas chromatography with tandem mass spectrometry) methodology was much less, equaling 45 and 34%. The pineapple swabbing experiment led to the detection of fludioxonil and diazinon below their respective maximum residue levels (MRLs), whereas measurements of untreated pineapple juice and other fruit extracts led to retrieval of dimethomorph, dinotefuran, imazalil, azoxystrobin, thiabendazole, fludioxonil, and diazinon, also below their MRL. The potential for mutual detection was investigated by mixing two standard solutions and by spiking an extract of fruit with a pesticide’s solution, and subsequently, individual compounds were simultaneously detected. For a selected subgroup of compounds, the bromide (Br-) chemical ionization characteristics were further inspected using quantum chemical computations to illustrate the structural features leading to their sensitive detection. Importantly, pesticides could be detected in actual extract and fruit samples, which demonstrates the potential of our fast screening method.Peer reviewe

Fractionation of Technical Lignin from Enzymatically Treated Steam-Exploded Poplar Using Ethanol and Formic Acid

Lignocellulosic biorefineries produce lignin-rich side streams with high valorization potential concealed behind their recalcitrant structure. Valorization of these residues to chemicals, materials, and fuels increases the profitability of biorefineries. Fractionation is required to reduce the lignins’ structural heterogeneity for further processing. We fractionated the technical biorefinery lignin received after steam explosion and saccharification processes. More homogeneous lignin fractions were produced with high β-O-4′ and aromatic content without residual carbohydrates. Non-toxic biodegradable organic solvents like ethanol and formic acid were used for fractionation and can be adapted to the existing biorefinery processes. Macromolecular properties of the isolated fractions were carefully characterized by structural, chemical, and thermal methods. The ethanol organosolv treatment produced highly soluble lignin with a reasonable yield, providing a uniform material for lignin applications. The organosolv fractionation with formic acid and combined ethanol-formic acid produced modified lignins that, based on thermal analysis, are promising as thermoresponsive materials.Lignocellulosic biorefineries produce lignin-rich side streams with high valorization potential concealed behind their recalcitrant structure. Valorization of these residues to chemicals, materials, and fuels increases the profitability of biorefineries. Fractionation is required to reduce the lignins' structural heterogeneity for further processing. We fractionated the technical biorefinery lignin received after steam explosion and saccharification processes. More homogeneous lignin fractions were produced with high beta-O-4 ' and aromatic content without residual carbohydrates. Non-toxic biodegradable organic solvents like ethanol and formic acid were used for fractionation and can be adapted to the existing biorefinery processes. Macromolecular properties of the isolated fractions were carefully characterized by structural, chemical, and thermal methods. The ethanol organosolv treatment produced highly soluble lignin with a reasonable yield, providing a uniform material for lignin applications. The organosolv fractionation with formic acid and combined ethanol-formic acid produced modified lignins that, based on thermal analysis, are promising as thermoresponsive materials.Peer reviewe

Exploring laccase mediator systems with acidic recombinant enzymes from Obba rivulosa

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Crystalline cyclophane-protein cage frameworks

open10siCyclophanes are macrocyclic supramolecular hosts famous for their ability to bind atomic or molecular guests via noncovalent interactions within their well-defined cavities. In a similar way, porous crystalline networks, such as metal organic frameworks, can create microenvironments that enable controlled guest binding in the solid state. Both types of materials often consist of synthetic components, and they have been developed within separate research fields. Moreover, the use of biomolecules as their structural units has remained elusive. Here, we have synthesized a library of organic cyclophanes and studied their electrostatic self-assembly with biological metal-binding protein cages (ferritins) into ordered structures. We show that cationic pillar[S]arenes and ferritin cages form biohybrid cocrystals with an open protein network structure. Our cyclophane-protein cage frameworks bridge the gap between molecular frameworks and colloidal nanoparticle crystals and combine the versatility of synthetic supramolecular hosts with the highly selective recognition properties of biomolecules. Such host-guest materials are interesting for porous material applications, including water remediation and heterogeneous catalysis.openBeyeh N.K.; Nonappa; Liljestrom V.; Mikkila J.; Korpi A.; Bochicchio D.; Pavan G.M.; Ikkala O.; Ras R.H.A.; Kostiainen M.A.Beyeh, N. K.; Nonappa, ; Liljestrom, V.; Mikkila, J.; Korpi, A.; Bochicchio, D.; Pavan, G. M.; Ikkala, O.; Ras, R. H. A.; Kostiainen, M. A

On the Effect of Hot-Water Pretreatment in Sulfur-Free Pulping of Aspen and Wheat Straw

Published under an ACS AuthorChoice LicenseIn modern biorefineries, low value lignin and hemicellulose fractions are produced as side streams. New extraction methods for their purification are needed in order to utilize the whole biomass more efficiently and to produce special target products. In several new applications using plant based biomaterials, the native-type chemical and polymeric properties are desired. Especially, production of high-quality native-type lignin enables valorization of biomass entirely, thus making novel processes sustainable and economically viable. To investigate sulfur-free possibilities for so-called "lignin first" technologies, we compared alkaline organosolv, formic acid organosolv, and ionic liquid processes to simple soda "cooking" using wheat straw and aspen as raw materials. All experiments were carried out using microwave-assisted pulping approach toy enable rapid heat transfer and convenient control of temperature and pressure. The main target was to evaluate the advantage of a brief hot water extraction as a pretreatment for the pulping process. Most of these novel pulping methods resulted in high-quality lignin, which may be valorized more diversely than kraft lignin. Lignin fractions were thoroughly analyzed with NMR (C-13 and HSQC) and gel permeation chromatography to study the quality of the collected lignin. The cellulose fractions were analyzed by determining their lignin contents and carbohydrate profiles for further utilization in cellulose-based products or biofuels.Peer reviewe

Proteiinihäkkeihin perustuvat biohybridimateriaalit

Nature-mimicking bottom-up approaches, such as molecular self-assembly and biotemplating, enable the preparation of highly organized objects with nanometer-scale precision. In addition, the utilization of biomacromolecules as building blocks or structure-directing agents in nanomaterials can lead to the development of highly biocompatible functional systems that can be prepared in mild and environmentally friendly conditions. This thesis demonstrates how functional and self-assembling biohybrid materials can be prepared by combining viruses and protein cages together with synthetic molecules, proteins and DNA origamis. In publication I amphiphilic Janus-dendrimers were synthesized and electrostatically co-assembled together with native Cowpea chlorotic mottle virus (CCMV) particles to study how the structure of different dendrimers affects the assembly process. The resulting complexes resembled naturally occurring inclusion bodies and could therefore benefit their research as a model system. In publication II a water-soluble phthalocyanine was combined with 1,3,6,8-pyrenetetrasulfonic acid and apoferritin protein cages to obtain photoactive crystals. The structure and size of the self-assembling crystals could be adjusted by changing the electrolyte concentration. Most importantly, the crystals were able to generate highly reactive singlet oxygen under irradiation of visible light and could therefore be utilized as an oxidizing agent. In publication III crystalline superlattices were prepared by combining different biomacromolecules. CCMV particles and avidin proteins were self-assembled into binary crystals that could be pre- or post-functionalized through interaction between avidin and different biotin-tagged functional groups such as enzymes, plasmonic gold nanoparticles and fluorescent dyes. In publication IV the tendency of CCMV capsid proteins (CP) to bind onto genetic material was utilized to coat DNA origami structures to obtain enhanced delivery of the origamis inside cells. Due to the fully programmable and therefore easily functionalized nature of DNA origamis, the presented method could be applied, for example, in the delivery of DNA origami conjugated compounds. Taken together, these studies show how multivalency, hydrophobicity, electrolyte concentration as well as particle size and shape affect the structure of self-assembling complexes and crystals. This information can be further applied when designing and creating new functional biohybrid materials.Luontoa imitoivat alhaalta ylös -lähestymistavat, kuten molekulaarinen itsejärjestäytyminen ja biotemplaatit, mahdollistavat erittäin hyvin järjestäytyneiden materiaalien valmistamisen nanometriluokan tarkkuudella. Lisäksi biomakromolekyylien hyödyntäminen joko rakennusosina tai rakennetta ohjaavina yksikköinä nanomateriaaleissa voi johtaa ympäristöystävällisin menetelmin valmistettavien funktionaalisten ja bioyhteensopivien systeemien kehittämiseen. Tässä väitöskirjassa osoitetaan, kuinka funktionaalisia ja itsejärjestäytyviä biohybridimateriaaleja voidaan valmistaa yhdistämällä viruksia ja proteiinihäkkejä synteettisten molekyylien, proteiinien ja DNA-origamien kanssa. Julkaisussa I syntetisoitiin lehmäpavun kloroosiläikkävirusten (CCMV) kanssa sähköstaattisesti järjestäytyviä amfifiilisiä Janus-dendrimeerejä, jotta voitiin tutkia kuinka dendrimeerien rakenne vaikuttaa järjestäytymisprosessiin. Muodostuneet kompleksit muistuttivat luonnossa esiintyviä inkluusiokappaleita ja niitä voitaisiinkin näin ollen hyödyntää mallisysteemeinä inkluusiokappaleiden tutkimuksessa. Julkaisussa II valmistettiin fotoaktiivisia kiteitä yhdistämällä vesiliukoista ftalosyaniinia, pyreeni-1,3,6,8-tetrasulfonihappoa ja apoferritiini proteiinihäkkejä. Itsejärjestäytyvien kiteiden rakennetta ja kokoa voitiin kontrolloida elektrolyyttikonsentraation avulla. Lisäksi säteilyttäessä kiteitä näkyvällä valolla, ne muodostivat erittäin reaktiivista singlettihappea, minkä vuoksi niitä voitaisiin käyttää hapettimina. Julkaisussa III valmistettiin kiteisiä hilarakenteita yhdistämällä erilaisia biomakromolekyylejä. CCMV partikkelit ja avidiini-proteiinit itsejärjestäytyivät binäärisiksi kiteiksi, jotka voitiin funktionalisoida etu- tai jälkikäteen avidiinin ja erilaisten biotiinilla merkittyjen funktionaalisten ryhmien, kuten entsyymien, plasmonisten kultananopartikkelien ja fluoresoivien väriaineiden, välisten vuorovaikutusten kautta. Julkaisussa IV hyödynnettiin CCMV:n kapsidiproteiinien (CP) kykyä sitoutua geneettiseen materiaaliin päällystämällä DNA-origami rakenteita CP:lla origamien tehostetun soluun kuljetuksen saavuttamiseksi. Koska DNA-origamit ovat täysin ohjelmoitavia ja siksi myös helposti funktionalisoitavia, voidaan esittämäämme kuljetusmenetelmää hyödyntää esimerkiksi DNA-origameihin kiinnitettävien aineiden kuljetuksessa. Yhteenvetona todettakoon, että yllä kuvatut tutkimukset osoittavat kuinka monivalenttisuus, hydrofobisuus, elektrolyyttikonsentraatio sekä partikkelien koko ja muoto vaikuttavat itsejärjestäytyvien kompleksien ja kiteiden rakenteeseen. Tätä tietoa voidaan jatkossa hyödyntää, kun suunnitellaan ja valmistetaan uusia funktionaalisia biohybridimateriaaleja

Kraft Process—Formation of Secoisolariciresinol Structures and Incorporation of Fatty Acids in Kraft Lignin

Correction to this article has published 4 May 2022: https://doi.org/10.1021/acs.jafc.2c02007The complex chemical structure and the fact that many areas in pulping and lignin chemistry still remain unresolved are challenges associated with exploiting lignin. In this study, we address questions regarding the formation and chemical nature of the insoluble residual lignin, the presence of fatty acids in kraft lignin, and the origin of secoisolariciresinol structures. A mild thermal treatment of lignin at maximum kraft-cooking temperatures (similar to 170 degrees C) with tall oil fatty acids (TOFA) or in an inert solvent (decane) produced highly insoluble products. However, acetylation of these samples enabled detailed chemical characterization by nuclear magnetic resonance (NMR) spectroscopy. The results show that the secoisolariciresinol (beta-beta) structure in kraft lignin is formed by rearrangement of the beta-aryl ether structure. Furthermore, fatty acids bind covalently to kraft lignin by reacting with the stilbene structures present. It is highly probable that these reactions also occur during kraft pulping, and this phenomenon has an impact on controlling the present kraft pulping process along with the development of new products from kraft lignin.Peer reviewe

Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins

Multicomponent crystals and nanoparticle superlattices are a powerful approach to integrate different materials into ordered nanostructures. Well-developed, especially DNA-based, methods for their preparation exist, yet most techniques concentrate on molecular and synthetic nanoparticle systems in non-biocompatible environment. Here we describe the self-assembly and characterization of binary solids that consist of crystalline arrays of native biomacromolecules. We electrostatically assembled cowpea chlorotic mottle virus particles and avidin proteins into heterogeneous crystals, where the virus particles adopt a non-close-packed body-centred cubic arrangement held together by avidin. Importantly, the whole preparation process takes place at room temperature in a mild aqueous medium allowing the processing of delicate biological building blocks into ordered structures with lattice constants in the nanometre range. Furthermore, the use of avidin–biotin interaction allows highly selective pre- or post-functionalization of the protein crystals in a modular way with different types of functional units, such as fluorescent dyes, enzymes and plasmonic nanoparticles.Peer reviewe