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- Item2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization(Frankfurt, M. : Beilstein-Institut zur Förderung 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.
- ItemA Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT(Weinheim : Wiley-VCH, 2021) Markel, Ulrich; Lanvers, Pia; Sauer, Daniel F.; Wittwer, Malte; Dhoke, Gaurao V.; Davari, Mehdi D.; Schiffels, Johannes; Schwaneberg, UlrichEnzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases. © 2020 The Authors. Published by Wiley-VCH GmbH
- ItemEngineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design(Weinheim : Wiley-VCH, 2020) Zhang, Lingling; Cui, Haiyang; Dhoke, Gaurao V.; Zou, Zhi; Sauer, Daniel F.; Davari, Mehdi D.; Schwaneberg, UlrichCopper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at −250 mV (up to 6.13 mA cm−2). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
- ItemOne‐Pot Two‐Step Chemoenzymatic Cascade for the Synthesis of a Bis‐benzofuran Derivative(Weinheim : Wiley-VCH Verl., 2019) Mertens, M.A. Stephanie; Thomas, Fabian; Nöth, Maximilian; Moegling, Julian; El‐Awaad, Islam; Sauer, Daniel F.; Dhoke, Gaurao V.; Xu, Wenjing; Pich, Andrij; Herres‐Pawlis, 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 84 % yield of the final product. The oxidation reaction is boosted by capturing inhibiting reaction components.
- 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 Joëlle; 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 μM) and a broad linear detection range (5 to 250 μM) 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). © 2018, The Author(s).
- ItemEngineered living hydrogels for robust biocatalysis in pure organic solvents(Maryland Heights, MO : Cell Press, 2022) Gao, Liang; Feng, Lilin; Sauer, Daniel F.; Wittwer, Malte; Hu, Yong; Schiffels, Johannes; Li, XinEngineered living hydrogels that can protect cells from harsh environments have achieved preliminary successes in biomedicine and environmental remediation. However, their biocatalytic applications in pure organic solvents have not been explored. Here, living hydrogels were engineered by integrating genetically modified Escherichia coli cells into alginate hydrogels for robust biocatalysis in pure organic solvents. The biocompatible hydrogels could not only support cell growth and diminish cell escape but could also act as protective matrices to improve organic solvent tolerance, thereby prolonging catalytic activity of whole-cell biocatalysts. Moreover, the influence of hydrogel microenvironments on biocatalytic efficiency was thoroughly investigated. Importantly, the versatility of engineered living hydrogels paves the way to achieve robust biocatalytic efficiency in a variety of pure organic co-solvents. Overall, we are able to engineer living hydrogels for regio-selective synthesis in pure organic solvents, which may be particularly useful for the innovation of living hydrogels in biocatalysis.