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Zugriffsart
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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.
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.
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.
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.
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.
Preprint: Studies on the enzymatic reduction of levulinic acid using Chiralidon-R and Chiralidon-S
(2023)
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.
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.
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.
Kartellrecht vs. Datenschutzrecht: Rechtsgrundlagen für die Datenverarbeitung in sozialen Netzwerken
(2023)
Umsatzbasierte Bußgelder – wie sonst nur aus dem Kartellrecht bekannt – waren einer der Gründe, warum die Datenschutz-Grundverordnung (DSGVO) vor ihrem Inkrafttreten für erhebliches Aufsehen sorgte. Die vielfach relevanteren Schadensersatzansprüche, die, wie bei „Dieselgate“, aufgrund der Vielzahl von betroffenen Personen und der aus Sicht von Rechtsdienstleistern bestehenden Skalierbarkeit mit weitaus höheren Einbußen für Unternehmen einhergehen können, blieben zunächst unbeachtet. Inzwischen ist der Schadensersatzanspruch gem. Art. 82 DSGVO die Vorschrift, die die meisten Vorlagen zum Europäischen Gerichtshof (EuGH) der letzten Jahre hervorgerufen hat. Am 4.5.2023 hat nun der EuGH (Urteil v. 4.5.2023 - Rs. C-300/21, NWB GAAAJ-41389) in einem Grundsatzurteil über zentrale Fragen rund um den Ersatz immaterieller Schäden als Folge von Datenschutzverstößen entschieden.
In dem vorliegenden Beitrag setzt sich der Verfasser mit dem Urteil des EuGH vom 4.5.2023 (Az.: C-60/22, DSB 2023, 178) zu den Auswirkungen eines formellen Verstoßes des Verantwortlichen gegen die Pflichten aus Artt. 26, 30 DSGVO (juris: EUV 2016/679) auf die Rechtmäßigkeit der Datenverarbeitung auseinander. Nachdem zunächst der zugrunde liegende Sachverhalt und der Hintergrund des Vorlageverfahrens skizziert wurden, gibt der Verfasser einen Überblick über die wesentlichen Entscheidungsgründe des EuGH. Insbesondere stelle der EuGH hier fest, dass die Rechtmäßigkeit der Verarbeitung in Art. 6 DSGVO geregelt sei und sich eine rechtswidrige Verarbeitung daher nur aus einem Verstoß gegen die Artt. 6 ff. DSGVO ergeben könne; die Pflichten aus Art. 26 und Art. 30 DSGVO würden nicht zu den Gründen für die Rechtmäßigkeit der Verarbeitung zählen. Mit Blick auf die Praxis lasse sich, so der Verfasser abschließend, festhalten, dass die Entscheidung insofern nicht überraschend sei; jedoch sei die Feststellung, dass sich aus Verstößen gegen Art. 26 und Art. 30 DSGVO kein Verstoß gegen das Grundrecht auf den Schutz personenbezogener Daten nachweisen lasse überraschend und bedenklich. Auch überrasche es, dass der EuGH eher in einem Nebensatz feststelle, dass der Verantwortliche im Prozess aufgrund seiner Rechenschaftspflicht gegenüber Betroffenen beweisbelastet ist; ob sich die Kammer hier der möglichen Auswirkungen ihrer Ausführungen bewusst gewesen sei, bleibe fraglich.
Nachhaltige Technologien, die Ressourcen schonen und Energie gewinnen, erlangen zunehmend an Bedeutung im urbanen Raum. Diese Bachelorarbeit befasst sich mit der Entwicklung eines Corporate Designs für ein Unternehmen, das sich auf die Fertigung und Planung von Bioenergiefassaden spezialisiert hat. Das Unternehmen sorgt durch seinen Fokus auf nachhaltige Energiegewinnung und effiziente Gebäudeplanung für die Verbesserung der ökologischen Herausforderungen. Das Ziel des neuen Corporate Designs ist es, die komplexe Thematik der Bioenergiefassaden der Zielgruppe effektiv zu vermitteln und ihr Interesse für dieses System zu wecken. Dabei werden Illustrationen und Infografiken eingesetzt, um die Technologie verständlich darzustellen, die positiven Umweltauswirkungen sowie Vorteile der Bioenergiefassaden deutlich hervorzuheben und mehr Umsetzungen zu erzielen.
In today's ultra-connected world, the significance of audio-visual documentation is rapidly growing. Technological advancements have made it more accessible to obtain powerful and compact audio-visual equipment, thereby reducing the dependency on a larger team for the documentation process. This has favoured the development of what is known as backpack journalism.
This project meets an unmet market need and presents an opportunity for innovative product development. It focuses on backpack journalists working in remote and challenging conditions with unpredictable transportation and working conditions. The proposed backpack design addresses both transport and working setups. Offering a robust and waterproof yet overall lightweight construction for easy equipment carrying, while also providing an extension of the workspace for essential gear.
The Cramér-von-Mises distance is applied to the distribution of the excess over a confidence level. Asymptotics of related statistics are investigated, and it is seen that the obtained limit distributions differ from the classical ones. For that reason, quantiles of the new limit distributions are given and new bootstrap techniques for approximation purposes are introduced and justified. The results motivate new one-sample goodness-of-fit tests for the distribution of the excess over a confidence level and a new confidence interval for the related fitting error. Simulation studies investigate size and power of the tests as well as coverage probabilities of the confidence interval in the finite sample case. A practice-oriented application of the Cramér-von-Mises tests is the determination of an appropriate confidence level for the fitting approach. The adoption of the idea to the well-known problem of threshold detection in the context of peaks over threshold modelling is sketched and illustrated by data examples.
Selected problems in the field of multivariate statistical analysis are treated. Thereby, one focus is on the paired sample case. Among other things, statistical testing problems of marginal homogeneity are under consideration. In detail, properties of Hotelling‘s T² test in a special parametric situation are obtained. Moreover, the nonparametric problem of marginal homogeneity is discussed on the basis of possibly incomplete data. In the bivariate data case, properties of the Hoeffding-Blum-Kiefer-Rosenblatt independence test statistic on the basis of partly not identically distributed data are investigated. Similar testing problems are treated within the scope of the application of a result for the empirical process of the concomitants for partly categorial data. Furthermore, testing changes in the modeled solvency capital requirement of an insurance company by means of a paired sample from an internal risk model is discussed. Beyond the paired sample case, a new asymptotic relative efficiency concept based on the expected volumes of multidimensional confidence regions is introduced. Besides, a new approach for the treatment of the multi-sample goodness-of-fit problem is presented. Finally, a consistent test for the treatment of the goodness-of-fit problem is developed for the background of huge or infinite dimensional data.
We consider time-dependent portfolios and discuss the allocation of changes in the risk of a portfolio to changes in the portfolio’s components. For this purpose we adopt established allocation principles. We also use our approach to obtain forecasts for changes in the risk of the portfolio’s components. To put the approach into practice we present an implementation based on the output of a simulation. Allocation is illustrated with an example portfolio in the context of Solvency II. The quality of the forecasts is investigated with an empirical study.
On the applicability of several tests to models with not identically distributed random effects
(2023)
We consider Kolmogorov–Smirnov and Cramér–von-Mises type tests for testing central symmetry, exchangeability, and independence. In the standard case, the tests are intended for the application to independent and identically distributed data with unknown distribution. The tests are available for multivariate data and bootstrap procedures are suitable to obtain critical values. We discuss the applicability of the tests to random effects models, where the random effects are independent but not necessarily identically distributed and with possibly unknown distributions. Theoretical results show the adequacy of the tests in this situation. The quality of the tests in models with random effects is investigated by simulations. Empirical results obtained confirm the theoretical findings. A real data example illustrates the application.
The feasibility study presents results of a hydrogen combustor integration for a Medium-Range aircraft engine using the Dry-Low-NOₓ Micromix combustion principle. Based on a simplified Airbus A320-type flight mission, a thermodynamic performance model of a kerosene and a hydrogen-powered V2530-A5 engine is used to derive the thermodynamic combustor boundary conditions. A new combustor design using the Dry-Low NOx Micromix principle is investigated by slice model CFD simulations of a single Micromix injector for design and off-design operation of the engine. Combustion characteristics show typical Micromix flame shapes and good combustion efficiencies for all flight mission operating points. Nitric oxide emissions are significant below ICAO CAEP/8 limits. For comparison of the Emission Index (EI) for NOₓ emissions between kerosene and hydrogen operation, an energy (kerosene) equivalent Emission Index is used.
A full 15° sector model CFD simulation of the combustion chamber with multiple Micromix injectors including inflow homogenization and dilution and cooling air flows investigates the combustor integration effects, resulting NOₓ emission and radial temperature distributions at the combustor outlet. The results show that the integration of a Micromix hydrogen combustor in actual aircraft engines is feasible and offers, besides CO₂ free combustion, a significant reduction of NOₓ emissions compared to kerosene operation.