TY - JOUR A1 - Heiden, W. A1 - Turek, M. A1 - Schöning, Michael Josef T1 - TasteIT : Analyzing chemical sensor data using fuzzy logic Y1 - 2011 SN - 978-1-4244-9910-6 N1 - 2011 IEEE Workshop on Merging Fields of Computational Intelligence and Sensor Technology ; 11.-15. April 2011 Paris, France SP - 1 EP - 6 PB - IEEE CY - New York ER - TY - JOUR A1 - Heiden, W. A1 - Turek, M. A1 - Schöning, Michael Josef T1 - Analysis of chemical sensor data JF - Proceedings of the 4th Russian-German Workshop "Innovation Information Technologies: Theory and practice" : Ufa, Russia, April 8-13, 2011 / eds. Yupsova, Nafisa ... Y1 - 2011 SN - 978-5-4221-0159-7 SP - 76 EP - 81 PB - State Aviation Technical Univ. CY - Ufa ER - TY - JOUR A1 - Handtke, Stefan A1 - Volland, Sonja A1 - Methling, Karen A1 - Albrecht, Dirk A1 - Becher, Dörte A1 - Nehls, Jenny A1 - Bongaerts, Johannes A1 - Maurer, Karl-Heinz A1 - Lalk, Michael A1 - Liesegang, Heiko A1 - Voigt, Birgit A1 - Daniel, Rolf A1 - Hecker, Michael T1 - Cell physiology of the biotechnological relevant bacterium Bacillus pumilus - An omics-based approach JF - Journal of Biotechnology N2 - Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC–MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed. Y1 - 2014 U6 - http://dx.doi.org/10.1016/j.jbiotec.2014.08.028 SN - 1873-4863 (E-Journal); 0168-1656 (Print) IS - 192(A) SP - 204 EP - 214 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Hamad, E. M. A1 - Bilatto, S. E. R. A1 - Adly, N. Y. A1 - Correa, D. S. A1 - Wolfrum, B. A1 - Schöning, Michael Josef A1 - Offenhäusser, A. A1 - Yakushenko, A. T1 - Inkjet printing of UV-curable adhesive and dielectric inks for microfluidic devices JF - Lab on a Chip N2 - Bonding of polymer-based microfluidics to polymer substrates still poses a challenge for Lab-On-a-Chip applications. Especially, when sensing elements are incorporated, patterned deposition of adhesives with curing at ambient conditions is required. Here, we demonstrate a fabrication method for fully printed microfluidic systems with sensing elements using inkjet and stereolithographic 3D-printing. Y1 - 2016 U6 - http://dx.doi.org/10.1039/C5LC01195G SN - 1473-0189 VL - 16 IS - 1 SP - 70 EP - 74 PB - Royal Society of Chemistry CY - Cambridge 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 - http://dx.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 - http://dx.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 - Jolmes, Tristan A1 - Oyen, Sven A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Schörken, Ulrich A1 - Siegert, Petra T1 - Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis JF - Applied Microbiology and Biotechnology N2 - N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR. KW - Chaperone KW - Biocatalysis KW - Aminoacylase KW - Acylation KW - Acyl-amino acids KW - Biosurfactants Y1 - 2024 U6 - http://dx.doi.org/10.1007/s00253-023-12868-8 SN - 1432-0614 N1 - Corresponding author: Petra Siegert IS - 108 PB - Springer CY - Berlin 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 - http://dx.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 - Bongaerts, Johannes A1 - Siegert, Petra T1 - A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent JF - Analytical Biochemistry N2 - An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates. Y1 - 2022 U6 - http://dx.doi.org/10.1016/j.ab.2022.114819 SN - 1096-0309 IS - 624 PB - Elsevier CY - Amsterdam 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 - JOUR A1 - Guo, Yuanyuan A1 - Seki, Kosuke A1 - Miyamoto, Ko-ichiro A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Yoshinobu, Tatsuo T1 - Novel photoexcitation method for light-addressable potentiometric sensor with higher spatial resolution JF - Applied physics express : APEX N2 - A novel photoexcitation method for the light-addressable potentiometric sensor (LAPS) is proposed to achieve a higher spatial resolution of chemical images. The proposed method employs a combined light source that consists of a modulated light probe, which generates the alternating photocurrent signal, and a ring of constant illumination surrounding it. The constant illumination generates a sheath of carriers with increased concentration which suppresses the spread of photocarriers by enhanced recombination. A device simulation was carried out to verify the effect of constant illumination on the spatial resolution, which demonstrated that a higher spatial resolution can be obtained. Y1 - 2014 U6 - http://dx.doi.org/10.7567/APEX.7.067301 SN - 1882-0786 (E-Journa); 1882-0778 (Print) VL - 7 IS - 6 SP - 067301-4 PB - IOP CY - Bristol ER - TY - JOUR A1 - Guo, Yuanyuan A1 - Miyamoto, Ko-ichiro A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Yoshinobu, Tatsuo T1 - Theoretical study and simulation of light-addressable potentiometric sensors JF - Physica status solidi (A) : applications and materials N2 - The light-addressable potentiometric sensor (LAPS) is a semiconductor-based potentiometric sensor using a light probe with an ability of detecting the concentration of biochemical species in a spatially resolved manner. As an important biomedical sensor, research has been conducted to improve its performance, for instance, to realize high-speed measurement. In this work, the idea of facilitating the device-level simulation, instead of using an equivalent-circuit model, is presented for detailed analysis and optimization of the performance of the LAPS. Both carrier distribution and photocurrent response have been simulated to provide new insight into both amplitude-mode and phase-mode operations of the LAPS. Various device parameters can be examined to effectively design and optimize the LAPS structures and setups for enhanced performance. Y1 - 2014 U6 - http://dx.doi.org/10.1002/pssa.201330354 SN - 0031-8965 VL - 211 IS - 6 SP - 1467 EP - 1472 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Guo, Yuanyuan A1 - Miyamoto, Ko-ichiro A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Yoshinobu, Tatsuo T1 - Device simulation of the light-addressable potentiometric sensor for the investigation of the spatial resolution JF - Sensors and actuators B: Chemical N2 - As a semiconductor-based electrochemical sensor, the light-addressable potentiometric sensor (LAPS) can realize two dimensional visualization of (bio-)chemical reactions at the sensor surface addressed by localized illumination. Thanks to this imaging capability, various applications in biochemical and biomedical fields are expected, for which the spatial resolution is critically significant. In this study, therefore, the spatial resolution of the LAPS was investigated in detail based on the device simulation. By calculating the spatiotemporal change of the distributions of electrons and holes inside the semiconductor layer in response to a modulated illumination, the photocurrent response as well as the spatial resolution was obtained as a function of various parameters such as the thickness of the Si substrate, the doping concentration, the wavelength and the intensity of illumination. The simulation results verified that both thinning the semiconductor substrate and increasing the doping concentration could improve the spatial resolution, which were in good agreement with known experimental results and theoretical analysis. More importantly, new findings of interests were also obtained. As for the dependence on the wavelength of illumination, it was found that the known dependence was not always the case. When the Si substrate was thick, a longer wavelength resulted in a higher spatial resolution which was known by experiments. When the Si substrate was thin, however, a longer wavelength of light resulted in a lower spatial resolution. This finding was explained as an effect of raised concentration of carriers, which reduced the thickness of the space charge region. The device simulation was found to be helpful to understand the relationship between the spatial resolution and device parameters, to understand the physics behind it, and to optimize the device structure and measurement conditions for realizing higher performance of chemical imaging systems. Y1 - 2014 U6 - http://dx.doi.org/10.1016/j.snb.2014.08.016 SN - 1873-3077 (E-Journal); 0925-4005 (Print) VL - 204 SP - 659 EP - 665 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Gun, Jenny A1 - Schöning, Michael Josef A1 - Abouzar, Maryam H. A1 - Poghossian, Arshak A1 - Katz, Evgeny T1 - Field-Effect Nanoparticle-Based Glucose Sensor on a Chip: Amplification Effect of Coimmobilized Redox Species JF - Electroanalysis. 20 (2008), H. 16 Y1 - 2008 SN - 1521-4109 SP - 1748 EP - 1753 ER - TY - JOUR A1 - Gun, Jenny A1 - Rizkov, Dan A1 - Lev, Ovadia A1 - Abouzar, Maryam H. A1 - Poghossian, Arshak A1 - Schöning, Michael Josef T1 - Oxygen plasma-treated gold nanoparticle-based field-effect devices as transducer structures for bio-chemical sensing JF - Microchimica Acta. 164 (2008), H. 3-4 Y1 - 2008 SN - 1436-5073 SP - 395 EP - 404 ER - TY - JOUR A1 - Gun, Jenny A1 - Gutkin, Vitaly A1 - Lev, Ovadia A1 - Boyen, Hans-Gerd A1 - Saitner, Marc A1 - Wagner, Patrick A1 - Olieslaeger, Marc D´ A1 - Abouzar, Maryam H. A1 - Poghossian, Arshak A1 - Schöning, Michael Josef T1 - Tracing gold nanoparticle charge by electrolyte-insulator-semiconductor devices JF - Journal of Physical Chemistry C. 115 (2011), H. 11 Y1 - 2011 SN - 1932-7455 SP - 4439 EP - 4445 PB - American Cemical Society CY - Washington, DC ER - TY - JOUR A1 - Grinsven, Bart van A1 - Bon, Natalie vanden A1 - Strauven, Hannelore A1 - Grieten, Lars A1 - Murib, Mohammed A1 - Jiménez Monroy, Kathia L. A1 - Janssens, Stoffel D. A1 - Haenen, Ken A1 - Schöning, Michael Josef A1 - Vermeeren, Veronique A1 - Ameloot, Marcel A1 - Michiels, Luc A1 - Thoelen, Ronald A1 - Ceuninck, Ward de A1 - Wagner, Patrick T1 - Heat-Transfer Resistance at Solid-Liquid Interfaces: A Tool for The Detection of Single Nucleotide Polymorphisms in DNA. JF - ACS Nano N2 - In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low- to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA. Y1 - 2012 U6 - http://dx.doi.org/10.1021/nn300147e SN - 1936-086X VL - 6 IS - 3 SP - 2712 EP - 2721 PB - ACS Publications CY - Washington, DC ER - TY - JOUR A1 - Grinsven, B. van A1 - Bon, N. vanden A1 - Grieten, L. A1 - Murib, M. A1 - Janssen, S. D. A1 - Haenen, K. A1 - Schneider, E. A1 - Ingebrandt, E. A1 - Schöning, Michael Josef A1 - Vermeeren, V. A1 - Ameloot, M. A1 - Michiels, L. A1 - Thoelen, R. A1 - Ceuninck, W. de A1 - Wagner, P. T1 - Rapid assessment of the stability of DNA duplexes by impedimetric real-time monitoring of chemically induced denaturation JF - Lab on a Chip Y1 - 2011 SN - 1473-0197 VL - 11 IS - 9 SP - 1656 EP - 1663 PB - Royal Society of Chemistry (RSC) CY - Cambridge ER - TY - JOUR A1 - Givanoudi, Stella A1 - Cornelis, Peter A1 - Rasschaert, Geertrui A1 - Wackers, Gideon A1 - Iken, Heiko A1 - Rolka, David A1 - Yongabi, Derick A1 - Robbens, Johan A1 - Schöning, Michael Josef A1 - Heyndrickx, Marc A1 - Wagner, Patrick T1 - Selective Campylobacter detection and quantification in poultry: A sensor tool for detecting the cause of a common zoonosis at its source JF - Sensors and Actuators B: Chemical Y1 - 2021 U6 - http://dx.doi.org/10.1016/j.snb.2021.129484 SN - 0925-4005 IS - In Press, Journal Pre-proof SP - Article 129484 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Ge, B. A1 - Meyer, T. A1 - Schöning, Michael Josef A1 - Wollenberger, U. A1 - Lisdat, F., T1 - Cytochrome c‘ from Chromatium vinosum on gold electrodes JF - Electrochemistry Communications. 2 (2000), H. 8 Y1 - 2000 SN - 1388-2481 SP - 557 EP - 561 ER -