TY - RPRT A1 - Haeger, Gerrit A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep) Y1 - 2023 N1 - Förderkennzeichen: 13FH256PA6 Titel: FHprofUnt 2016: Neue biobasierte Lipopeptide aus nachhaltiger Produktion Laufzeit: 01.02.2019 – 31.10.2022 ER - TY - JOUR A1 - Haeger, Gerrit A1 - Grankin, Alina A1 - Wagner, Michaela T1 - Construction of an Aspergillus oryzae triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology JF - Applied Research N2 - Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems. KW - aspergillus KW - CRISPR/Cas9 KW - filamentous fungi KW - genome engineering Y1 - 2023 U6 - https://doi.org/10.1002/appl.202200106 SN - 2702-4288 IS - Early View SP - 1 EP - 15 PB - Wiley-VCH ER - TY - JOUR A1 - Haeger, Gerrit A1 - Probst, Johanna A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans JF - FEBS Open Bio N2 - Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl-amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236ᵀ . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC 3.5.1.14), designated SgAA, and an ε-lysine acylase (EC 3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl-amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl-amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-L-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl-amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine. KW - Streptomyces lividans KW - recombinant expression KW - Streptomyces griseus KW - ε-lysine acylase KW - α-aminoacylase Y1 - 2023 U6 - https://doi.org/10.1002/2211-5463.13723 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 12 SP - 2224 EP - 2238 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Haeger, Gerrit A1 - Wirges, Jessika A1 - Tanzmann, Nicole A1 - Oyen, Sven A1 - Jolmes, Tristan A1 - Jaeger, Karl-Erich A1 - Schörken, Ulrich A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis JF - Microbial Cell Factories N2 - Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated. KW - Acyl-amino acids KW - Inclusion bodies KW - Chaperone co-expression KW - Vibrio natriegens KW - Aminoacylase Y1 - 2023 U6 - https://doi.org/10.1186/s12934-023-02079-1 SN - 1475-2859 N1 - Corresponding author: Petra Siegert IS - 22 SP - Article number: 77 (2023) PB - Springer Nature ER - TY - JOUR A1 - Hammer, Thorben A1 - Quitter, Julius A1 - Mayntz, Joscha A1 - Bauschat, J.-Michael A1 - Dahmann, Peter A1 - Götten, Falk A1 - Hille, Sebastian A1 - Stumpf, Eike T1 - Free fall drag estimation of small-scale multirotor unmanned aircraft systems using computational fluid dynamics and wind tunnel experiments JF - CEAS Aeronautical Journal N2 - New European Union (EU) regulations for UAS operations require an operational risk analysis, which includes an estimation of the potential danger of the UAS crashing. A key parameter for the potential ground risk is the kinetic impact energy of the UAS. The kinetic energy depends on the impact velocity of the UAS and, therefore, on the aerodynamic drag and the weight during free fall. Hence, estimating the impact energy of a UAS requires an accurate drag estimation of the UAS in that state. The paper at hand presents the aerodynamic drag estimation of small-scale multirotor UAS. Multirotor UAS of various sizes and configurations were analysed with a fully unsteady Reynolds-averaged Navier–Stokes approach. These simulations included different velocities and various fuselage pitch angles of the UAS. The results were compared against force measurements performed in a subsonic wind tunnel and provided good consistency. Furthermore, the influence of the UAS`s fuselage pitch angle as well as the influence of fixed and free spinning propellers on the aerodynamic drag was analysed. Free spinning propellers may increase the drag by up to 110%, depending on the fuselage pitch angle. Increasing the fuselage pitch angle of the UAS lowers the drag by 40% up to 85%, depending on the UAS. The data presented in this paper allow for increased accuracy of ground risk assessments. KW - Multirotor UAS KW - Drag estimation KW - CFD KW - Wind tunnel experiments KW - Wind milling Y1 - 2023 U6 - https://doi.org/10.1007/s13272-023-00702-w SN - 1869-5590 (Online) SN - 1869-5582 (Print) N1 - Corresponding author: Thorben Hammer PB - Springer CY - Wien ER - TY - THES A1 - Hatke, Lars T1 - X Plore: human hybrid submarine N2 - Das „X Plore“ ist ein human hybrid angetriebenes Tauchboot, welches sowohl für den touristischen als auch privaten Gebrauch konzipiert ist. Das immersive Design des „X Plore" sorgt für ein eindrucksvolles Taucherlebnis. Das Konzept soll die Menschen über die aktuelle Not der Meere und ihrer Lebewesen aufklären und ihnen gleichzeitig die Möglichkeit geben, der Unterwasserwelt näher zukommen. Zudem soll es diese erhalten und ihre Bewohner:innen schützen. Des Weiteren thematisiert die Arbeit zukunftsorientierte Antriebs- und Batterietechniken und setzt sich mit der sinnvollen Integrierung dieser auseinander. KW - Entdecken KW - Abenteuer KW - Erkunden KW - Unterwasserwelt KW - Tauchboot Y1 - 2023 N1 - Für diese Arbeit steht kein Volltext zur Verfügung. PB - FH Aachen CY - Aachen ER - TY - JOUR A1 - Heieis, Jule A1 - Böcker, Jonas A1 - D'Angelo, Olfa A1 - Mittag, Uwe A1 - Albracht, Kirsten A1 - Schönau, Eckhard A1 - Meyer, Andreas A1 - Voigtmann, Thomas A1 - Rittweger, Jörn T1 - Curvature of gastrocnemius muscle fascicles as function of muscle–tendon complex length and contraction in humans JF - Physiological Reports N2 - It has been shown that muscle fascicle curvature increases with increasing contraction level and decreasing muscle–tendon complex length. The analyses were done with limited examination windows concerning contraction level, muscle–tendon complex length, and/or intramuscular position of ultrasound imaging. With this study we aimed to investigate the correlation between fascicle arching and contraction, muscle–tendon complex length and their associated architectural parameters in gastrocnemius muscles to develop hypotheses concerning the fundamental mechanism of fascicle curving. Twelve participants were tested in five different positions (90°/105°*, 90°/90°*, 135°/90°*, 170°/90°*, and 170°/75°*; *knee/ankle angle). They performed isometric contractions at four different contraction levels (5%, 25%, 50%, and 75% of maximum voluntary contraction) in each position. Panoramic ultrasound images of gastrocnemius muscles were collected at rest and during constant contraction. Aponeuroses and fascicles were tracked in all ultrasound images and the parameters fascicle curvature, muscle–tendon complex strain, contraction level, pennation angle, fascicle length, fascicle strain, intramuscular position, sex and age group were analyzed by linear mixed effect models. Mean fascicle curvature of the medial gastrocnemius increased with contraction level (+5 m−1 from 0% to 100%; p = 0.006). Muscle–tendon complex length had no significant impact on mean fascicle curvature. Mean pennation angle (2.2 m−1 per 10°; p < 0.001), inverse mean fascicle length (20 m−1 per cm−1; p = 0.003), and mean fascicle strain (−0.07 m−1 per +10%; p = 0.004) correlated with mean fascicle curvature. Evidence has also been found for intermuscular, intramuscular, and sex-specific intramuscular differences of fascicle curving. Pennation angle and the inverse fascicle length show the highest predictive capacities for fascicle curving. Due to the strong correlations between pennation angle and fascicle curvature and the intramuscular pattern of curving we suggest for future studies to examine correlations between fascicle curvature and intramuscular fluid pressure. KW - biomechanics KW - connective tissue KW - physiology KW - ultrasound Y1 - 2023 U6 - https://doi.org/10.14814/phy2.15739 SN - 2051-817X VL - 11 IS - 11 SP - e15739, Seite 1-11 PB - Wiley ER - TY - CHAP A1 - Heimes, Heiner Hans A1 - Kampker, Achim A1 - Dorn, Benjamin A1 - Kehrer, Mario A1 - Dünnwald, Simon A1 - Badura, Dennis A1 - Terren, Maximilian A1 - Röth, Thilo ED - Kampker, Achim ED - Heimes, Heiner Hans T1 - Produktionsprozesse der Fahrzeugstruktur T2 - Elektromobilität: Grundlagen einer Fortschrittstechnologie Y1 - 2023 SN - 978-3-662-65811-6 (Print) SN - 978-3-662-65812-3 (Online) U6 - https://doi.org/10.1007/978-3-662-65812-3_13 N1 - Corresponding author: Benjamin Dorn SP - 227 EP - 247 PB - Springer Vieweg CY - Berlin ER - TY - CHAP A1 - Heimes, Heiner Hans A1 - Kampker, Achim A1 - Kehrer, Mario A1 - Dünnwald, Simon A1 - Heetfeld, Lennart A1 - Polzenberg, Jens A1 - Budde, Lucas A1 - Keusen, Maximilian A1 - Pandey, Rahul A1 - Röth, Thilo ED - Kampker, Achim ED - Heimes, Heiner Hans T1 - Fahrzeugstruktur T2 - Elektromobilität: Grundlagen einer Fortschrittstechnologie N2 - Um sowohl Treibhausgas-Emissionen zu verringern als auch Kraftstoffressourcen zu schonen, wird zunehmend an einer Transformation konventionell angetriebener Kraftfahrzeuge hin zu elektrifizierten Antriebskonzepten gearbeitet. Basierend auf herkömmlichen Fahrzeugen mit Verbrennungsmotor wurde eine Vielzahl neuer Antriebssysteme mit verschiedenem Elektrifizierungsgrad entwickelt. Mitte der 1990er-Jahre kamen erste Fahrzeuge mit einem Hybridantrieb auf den Markt. Die Kombination aus Verbrennungs- und Elektromotor erlaubt eine Verbrauchsreduktion und Bremsenergierückgewinnung sowie lokal emissionsfreies Fahren. Y1 - 2023 SN - 978-3-662-65811-6 (Print) SN - 978-3-662-65812-3 (Online) U6 - https://doi.org/10.1007/978-3-662-65812-3_5 N1 - Corresponding author: Heiner Hans Heimes SP - 69 EP - 106 PB - Springer Vieweg CY - Berlin ER - TY - THES A1 - Heindrichs, Maik T1 - Intrinsic Motion in Type : eine Bewegtbild Plakatreihe für das Düsseldorfer Schauspielhaus N2 - Gestaltung und Programmierung dynamischer Motion-Design Plakate zwecks Kommunikation der Theaterstücke des Düsseldorfer Schauspielhauses. Der Fokus liegt dabei auf der Typografie und ihrer intrinsischen Bewegung. Der technische Fortschritt bietet viele neue Möglichkeiten im Umgang mit Schrift. Diese Arbeit dient dazu, mithilfe von aktuellen Techniken einen neuen Ansatz im Bereich Gestaltung mit Schrift, sowie Variable Fonts, in Kombination mit Motion-Design aufzuzeigen. Dies wird anhand von Eigenschaften der Schrift und der inhaltlichen Ebene, ohne die Buchstaben dabei zu dekonstruieren, umgesetzt. Die Bewegungen werden dann in das Format Plakat übertragen und dargestellt. KW - Bewegtbild KW - Plakat KW - Animation KW - Variable Schriften KW - Schriftvarianten Y1 - 2023 N1 - Für diese Arbeit steht kein Volltext zur Verfügung. PB - FH Aachen CY - Aachen ER -