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- ItemTurning a Killing Mechanism into an Adhesion and Antifouling Advantage(Weinheim : Wiley-VCH, 2019) Dedisch, Sarah; Obstals, Fabian; los Santos Pereira, Andres; Bruns, Michael; Jakob, Felix; Schwaneberg, Ulrich; RodriguezāEmmenegger, CesarMild and universal methods to introduce functionality in polymeric surfaces remain a challenge. Herein, a bacterial killing mechanism based on amphiphilic antimicrobial peptides is turned into an adhesion advantage. Surface activity (surfactant) of the antimicrobial liquid chromatography peak I (LCI) peptide is exploited to achieve irreversible binding of a proteināpolymer hybrid to surfaces via physical interactions. The proteināpolymer hybrid consists of two blocks, a surface-affine block (LCI) and a functional block to prevent protein fouling on surfaces by grafting antifouling polymers via single electron transfer-living radical polymerization (SET-LRP). The mild conditions of SET-LRP of N-2-hydroxy propyl methacrylamide (HPMA) and carboxybetaine methacrylamide (CBMAA) preserve the secondary structure of the fusion protein. Adsorption kinetics and grafting densities are assessed using surface plasmon resonance and ellipsometry on model gold surfaces, while the functionalization of a range of artificial and natural surfaces, including teeth, is directly observed by confocal microscopy. Notably, the fusion protein modified with poly(HPMA) completely prevents the fouling from human blood plasma and thereby exhibits a resistance to protein fouling that is comparable to the best grafted-from polymer brushes. This, combined with their simple application on a large variety of materials, highlights the universal and scalable character of the antifouling concept. Ā© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemAre Directed Evolution Approaches Efficient in Exploring Natureās Potential to Stabilize a Lipase in Organic Cosolvents?(Basel : MDPI, 2017) Markel, Ulrich; Zhu, Leilei; Frauenkron-Machedjou, Victorine; Zhao, Jing; Bocola, Marco; Davari, Mehdi; Jaeger, Karl-Erich; Schwaneberg, UlrichDespite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) libraryācovering the full natural diversity of single amino acid substitutions at all 181 positions of BSLAāwith three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzymeās resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%ā11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%ā12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistanceāmost likely due to a stronger attraction of water molecules. Ā© 2017 by the authors.
- ItemCorrection: A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels(Cambridge : Soc., 2015) LĆ¼lsdorf, Nina; Pitzler, Christian; Biggel, Michael; Martinez, Ronny; Vojcic, Ljubica; Schwaneberg, UlrichCorrection for āA flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogelsā by Nina LĆ¼lsdorf et al., Chem. Commun., 2015, 51, 8679ā8682.
- Item2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of Ć-barrel hybrid catalyst for phenylacetylene polymerization(Frankfurt, M. : Beilstein-Institut zur Fƶrderung der Chemischen Wissenschaften, 2017) Kinzel, Julia; Sauer, Daniel F.; Bocola, Marco; Arlt, Marcus; Mirzaei Garakani, Tayebeh; Thiel, Andreas; Beckerle, Klaus; Polen, Tino; Okuda, Jun; Schwaneberg, UlrichCovering hydrophobic regions with stabilization agents to solubilize purified transmembrane proteins is crucial for their application in aqueous media. The small molecule 2-methyl-2,4-pentanediol (MPD) was used to stabilize the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA) utilized as host for the construction of a rhodium-based biohybrid catalyst. Unlike commonly used detergents such as sodium dodecyl sulfate or polyethylene polyethyleneglycol, MPD does not form micelles in solution. Molecular dynamics simulations revealed the effect and position of stabilizing MPD molecules. The advantage of the amphiphilic MPD over micelle-forming detergents is demonstrated in the polymerization of phenylacetylene, showing a ten-fold increase in yield and increased molecular weights.
- ItemBiadhesive Peptides for Assembling Stainless Steel and Compound Loaded Micro-Containers(Weinheim : Wiley-VCH, 2019) Apitius, Lina; Buschmann, Sven; Bergs, Christian; Schƶnauer, David; Jakob, Felix; Pich, Andrij; Schwaneberg, UlrichBiadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materialsāfor example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells.
- ItemOneāPot TwoāStep Chemoenzymatic Cascade for the Synthesis of a Bisābenzofuran Derivative(Weinheim : Wiley-VCH Verl., 2019) Mertens, M.A. Stephanie; Thomas, Fabian; Nƶth, Maximilian; Moegling, Julian; ElāAwaad, Islam; Sauer, Daniel F.; Dhoke, Gaurao V.; Xu, Wenjing; Pich, Andrij; HerresāPawlis, Sonja; Schwaneberg, UlrichChemoenzymatic cascades enable reactions with the high productivity of chemocatalysts and high selectivity of enzymes. Nevertheless, the combination of these different fields of catalysis is prone to mutual deactivation of metal- and biocatalysts. In this study, a one-pot sequential two-step catalytic cascade reaction was successfully implemented for the synthesis of a methylene-bridged bis(2-substituted benzofuran). In the first step, a palladium-free Sonogashira reaction is used for the synthesis of a benzofuran derivative. In the subsequent step, the formed 2-substituted benzofuran is hydroxylated by the monooxygenase P450 BM3 variant (A74S-F87V-L188Q) and undergoes further elimination reactions. The study proofs that combination of Cu scorpionate catalyzed Sonogashira cross-coupling and P450 mediated oxidation is possible and results in up to 84ā% yield of the final product. The oxidation reaction is boosted by capturing inhibiting reaction components.
- ItemDirected Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles(Basel : Molecular Diversity Preservation International (MDPI), 2019) Santos, Gustavo de Almeida; Dhoke, Gaurao V.; Davari, Mehdi D.; Ruff, Anna JoĆ«lle; Schwaneberg, UlrichThe O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ā140-fold (WT: 8.3 Ā± 1.3 minā1; R255L: 1168 Ā± 163 minā1), total turnover number (TTN) increased ā21-fold (WT: 40 Ā± 3; R255L: 860 Ā± 15), and coupling efficiency, ā2.9-fold (WT: 8.8 Ā± 0.1%; R255L: 25.7 Ā± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.
- ItemDisulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability(Basel : Molecular Diversity Preservation International (MDPI), 2019) Bashirova, Anna; Pramanik, Subrata; Volkov, Pavel; Rozhkova, Aleksandra; Nemashkalov, Vitaly; Zorov, Ivan; Gusakov, Alexander; Sinitsyn, Arkady; Schwaneberg, Ulrich; Davari, Mehdi D.Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15ā21% increase in specific activity against carboxymethylcellulose and Ī²-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 Ā°C for 2 h, they retained 52ā58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 Ā°C, the enzyme-engineered forms retained 15ā22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
- ItemA hydroquinone-specific screening system for directed P450 evolution(Berlin ; Heidelberg ; New York : Springer, 2018) Weingartner, Alexandra M.; Sauer, Daniel F.; Dhoke, Gaurao V.; Davari, Mehdi D.; Ruff, Anna JoĆ«lle; Schwaneberg, UlrichThe direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 Ī¼M) and a broad linear detection range (5 to 250 Ī¼M) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). Ā© 2018, The Author(s).
- ItemTargeting microplastic particles in the void of diluted suspensions(Amsterdam [u.a.] : Elsevier Science, 2019) Islam, Shohana; Apitius, Lina; Jakob, Felix; Schwaneberg, UlrichAccumulation of microplastic in the environment and food chain will be a grand challenge for our society. Polyurethanes are widely used synthetic polymers in medical (e.g. catheters) and industrial products (especially as foams). Polyurethane is not abundant in nature and only a few microbial strains (fungi and bacteria) and enzymes (polyurethaneases and cutinases) have been reported to efficiently degrade polyurethane. Notably, in nature a long period of time (from 50 to >100āÆyears depending on the literature) is required for degradation of plastics. Material binding peptides (e.g. anchor peptides) bind strongly to polymers such as polypropylene, polyethylene terephthalate, and polyurethane and can target specifically polymers. In this study we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278 which accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278. Additionally, degradation half-lives of polyester-polyurethane nanoparticles were reduced from 41.8āÆh to 6.2āÆh (6.7-fold) in a diluted polyester-polyurethane suspension (0.04% w/v).