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 - 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 - 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 - 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 - THES A1 - Götz, Daniela T1 - Entwicklung eines Erscheinungsbildes für das Start-up-Unternehmen CoArea N2 - Diese Bachelorarbeit befasst sich mit dem Corporate Design des aufstrebenden Startups "CoArea". Seine innovative Plattform vermittelt ungenutzte Flächen im urbanen Raum und bringt Flächenbedarf und -bereitstellung intelligent zusammen. Die Arbeit untersucht, wie das Erscheinungsbild von CoArea das Unternehmen als modern, nachhaltig und ressourcenschonend positioniert. Durch das neu gestaltete Corporate Design wird diese besondere Herangehensweise zur effizienten Nutzung begrenzter Ressourcen unserer Umwelt durch CoArea deutlich und nach außen getragen. CoArea strebt danach, die positive Entwicklung der Städte voranzutreiben und die soziale Nachhaltigkeit durch den Austausch und die Interaktion zwischen verschiedenen Menschen und Gemeinschaften zu fördern. KW - Start-up KW - Nachhaltigkeit KW - Flächennutzung KW - Schraffur KW - Ressourcenschonung Y1 - 2023 PB - FH Aachen CY - Aachen ER - TY - CHAP A1 - Grund, Raphael M. A1 - Altherr, Lena ED - Reiff-Stephan, Jörg ED - Jäkel, Jens ED - Schwarz, André T1 - Development of an open source energy disaggregation tool for the home automation platform Home Assistant T2 - Tagungsband AALE 2023 : mit Automatisierung gegen den Klimawandel N2 - In order to reduce energy consumption of homes, it is important to make transparent which devices consume how much energy. However, power consumption is often only monitored aggregated at the house energy meter. Disaggregating this power consumption into the contributions of individual devices can be achieved using Machine Learning. Our work aims at making state of the art disaggregation algorithms accessibe for users of the open source home automation platform Home Assistant. KW - Home Automation Platform KW - Home Assistant KW - Open Source KW - Machine Learning KW - Energy Disaggregation Y1 - 2023 SN - 978-3-910103-01-6 U6 - https://doi.org/10.33968/2023.02 N1 - 19. AALE-Konferenz. Luxemburg, 08.03.-10.03.2023. BTS Connected Buildings & Cities Luxemburg (Tagungsband unter https://doi.org/10.33968/2023.01) SP - 11 EP - 20 PB - le-tex publishing services GmbH CY - Leipzig ER - TY - INPR A1 - Greiner, Lasse A1 - Jeromin, Günter Erich A1 - Sithole, Patience A1 - Petersen, Soenke T1 - Preprint: Studies on the enzymatic reduction of levulinic acid using Chiralidon-R and Chiralidon-S T2 - ChemRxiv N2 - The enzymatic reduction of levulinic acid by the chiral catalysts Chiralidon-R and Chiralidon-S which are commercially available superabsorbed alcohol dehydrogenases is described. The Chiralidon®-R/S reduces the levulinic acid to the (R,S)-4-hydroxy valeric acid and the (R)- or (S)- gamma-valerolactone. KW - Levulinic acid KW - Chiralidon-R KW - Chiralidon-S KW - 4-hydroxy valeric acid KW - (R)- or (S)- gamma-valerolactone Y1 - 2023 U6 - https://doi.org/10.26434/chemrxiv-2023-jlvcv ER - TY - JOUR A1 - Grajewski, Matthias A1 - Kleefeld, Andreas T1 - Detecting and approximating decision boundaries in low-dimensional spaces JF - Numerical Algorithms N2 - A method for detecting and approximating fault lines or surfaces, respectively, or decision curves in two and three dimensions with guaranteed accuracy is presented. Reformulated as a classification problem, our method starts from a set of scattered points along with the corresponding classification algorithm to construct a representation of a decision curve by points with prescribed maximal distance to the true decision curve. Hereby, our algorithm ensures that the representing point set covers the decision curve in its entire extent and features local refinement based on the geometric properties of the decision curve. We demonstrate applications of our method to problems related to the detection of faults, to multi-criteria decision aid and, in combination with Kirsch’s factorization method, to solving an inverse acoustic scattering problem. In all applications we considered in this work, our method requires significantly less pointwise classifications than previously employed algorithms. KW - MCDA KW - Inverse scattering problem KW - Fault approximation KW - Fault detection Y1 - 2023 SN - 1572-9265 N1 - Corresponding author: Matthias Grajewski VL - 93 IS - 4 PB - Springer Science+Business Media CY - Dordrecht ER - TY - JOUR A1 - Golland, Alexander A1 - Kriegesmann, Torben T1 - Zivilprozessuale Fragen zum datenschutzrechtlichen Schadensersatzanspruch JF - MMR - Zeitschrift für IT-Recht und Recht der Digitalisierung N2 - Im Verfahren gegen die Österreichische Post AG (Rs. C-300/21) befasste sich der EuGH erstmals mit dem in Art. 82 DS-GVO geregelten datenschutzrechtlichen Schadensersatzanspruch. Mit den Klarstellungen des EuGH verschieben sich die Probleme nun stärker zu den „klassischen“ Fragen des Schadensersatzrechts im Zivilprozess. Relevant sind dabei vor allem Aspekte der Darlegungs- und Beweislast und deren Besonderheiten mit Blick auf den Ersatz immaterieller Schäden. Der Beitrag fokussiert sich auf die Voraussetzungen und den dabei zu führenden Tatsachenbeweis bei der Klage des Betroffenen gegen den Verantwortlichen auf Ersatz immaterieller Schäden. Y1 - 2023 SN - 2698-7988 IS - 10 SP - 733 EP - 739 PB - Beck CY - München ER - TY - JOUR A1 - Golland, Alexander A1 - Kelbch, Niklas T1 - Kartellrecht vs. Datenschutzrecht: Rechtsgrundlagen für die Datenverarbeitung in sozialen Netzwerken JF - DSB Datenschutz-Berater Y1 - 2023 SN - 0170-7256 IS - 9 SP - 247 EP - 249 PB - DFV Mediengruppe CY - Frankfurt a.M. ER -