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DDC: 540 × Type: article × Publisher: Basel : MDPI AG × WGL Institute: DWI ×

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    Effects of proline substitutions on the thermostable LOV domain from Chloroflexus aggregans
    (Basel : MDPI AG, 2020) Remeeva, Alina; Nazarenko, Vera V.; Goncharov, Ivan M.; Yudenko, Anna; Smolentseva, Anastasia; Semenov, Oleg; Kovalev, Kirill; Gülbahar, Cansu; Schwaneberg, Ulrich; Davari, Mehdi D.; Gordeliy, Valentin; Gushchin, Ivan
    Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2◦ C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
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    Engineering robust cellulases for tailored lignocellulosic degradation cocktails
    (Basel : MDPI AG, 2020) Contreras, Francisca; Pramanik, Subrata; Rozhkova, Aleksandra M.; Zorov, Ivan N.; Korotkova, Olga; Sinitsyn, Arkady P.; Schwaneberg, Ulrich; Davari, Mehdi D.
    Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
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    Oligoglycidol-functionalised styrene macromolecules as reactive surfactants in the emulsion polymerisation of styrene: The impact of chain length and concentration on particle size and colloidal stability
    (Basel : MDPI AG, 2020) Waulthers, Kim; van Zandvoort, Ryan; Castermans, Sam; Welzen, Jeroen; Baeten, Evelien; Stout, Kathleen; Keul, Helmut; Mann, Daniel; Buskens, Pascal
    Reactive surfactants (surfmers), which are covalently attached to the surface of sub-micron sized polymer particles during emulsion polymerisation, are applied to tailor the surface functionality of polymer particles for an application of choice. We present a systematic study on the use of oligoglycidol-functionalised styrene macromolecules as surfmers in the emulsion polymerization of styrene. Firstly, we report the impact of the surfmer concentration on the particle size for polymerisations performed above and below the critical micelle concentration. Secondly, we report the influence of the oligoglycidol chain length on the particle size. Thirdly, we conducted experiments to analyse the influence of the surfmer concentration and its chain length on the colloidal stability of the aqueous polystyrene nanoparticles in sodium chloride solutions. We demonstrated that the size of polystyrene particles could be influenced by changing both the surfmer concentration and its chain length. Furthermore, we showed that the colloidal stability of the oligoglycidol-functionalized polystyrene particles is dependent on the particle size, and not directly related to the oligoglycidol chain length. © 2020 by the authors.