@article{HaegerJolmesOyenetal.2024, author = {Haeger, Gerrit and Jolmes, Tristan and Oyen, Sven and Jaeger, Karl-Erich and Bongaerts, Johannes and Sch{\"o}rken, Ulrich and Siegert, Petra}, title = {Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis}, series = {Applied Microbiology and Biotechnology}, journal = {Applied Microbiology and Biotechnology}, number = {108}, publisher = {Springer}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12868-8}, pages = {14 Seiten}, year = {2024}, abstract = {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.}, language = {en} } @article{KarschuckSchmidtAchtsnichtetal.2023, author = {Karschuck, Tobias and Schmidt, Stefan and Achtsnicht, Stefan and Poghossian, Arshak and Wagner, Patrick and Sch{\"o}ning, Michael Josef}, title = {Multiplexing system for automated characterization of a capacitive field-effect sensor array}, series = {Physica Status Solidi A}, volume = {220}, journal = {Physica Status Solidi A}, number = {22}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6300 (Print)}, doi = {10.1002/pssa.202300265}, pages = {7 Seiten}, year = {2023}, abstract = {In comparison to single-analyte devices, multiplexed systems for a multianalyte detection offer a reduced assay time and sample volume, low cost, and high throughput. Herein, a multiplexing platform for an automated quasi-simultaneous characterization of multiple (up to 16) capacitive field-effect sensors by the capacitive-voltage (C-V) and the constant-capacitance (ConCap) mode is presented. The sensors are mounted in a newly designed multicell arrangement with one common reference electrode and are electrically connected to the impedance analyzer via the base station. A Python script for the automated characterization of the sensors executes the user-defined measurement protocol. The developed multiplexing system is tested for pH measurements and the label-free detection of ligand-stabilized, charged gold nanoparticles.}, language = {en} } @article{JanusAchtsnichtDrinicetal.2023, author = {Janus, Kevin Alexander and Achtsnicht, Stefan and Drinic, Aleksander and Kopp, Alexander and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Transient magnesium-based thin-film temperature sensor on a flexible, bioabsorbable substrate for future medical applications}, series = {Applied Research}, journal = {Applied Research}, number = {Accepted manuscript}, publisher = {Wiley-VCH}, issn = {2702-4288 (Print)}, doi = {10.1002/appl.202300102}, pages = {22 Seiten}, year = {2023}, abstract = {In this work, the bioabsorbable materials, namely fibroin, polylactide acid (PLA), magnesium and magnesium oxide are investigated for their application as transient, resistive temperature detectors (RTD). For this purpose, a thin-film magnesium-based meander-like electrode is deposited onto a flexible, bioabsorbable substrate (fibroin or PLA) and encapsulated (passivated) by additional magnesium oxide layers on top and below the magnesium-based electrode. The morphology of different layered RTDs is analyzed by scanning electron microscopy. The sensor performance and lifetime of the RTD is characterized both under ambient atmospheric conditions between 30°C and 43°C, and wet tissue-like conditions with a constant temperature regime of 37°C. The latter triggers the degradation process of the magnesium-based layers. The 3-layers RTDs on a PLA substrate could achieve a lifetime of 8.5 h. These sensors also show the best sensor performance under ambient atmospheric conditions with a mean sensitivity of 0.48 Ω/°C ± 0.01 Ω/°C.}, language = {en} } @article{HaegerProbstJaegeretal.2023, author = {Haeger, Gerrit and Probst, Johanna and Jaeger, Karl-Erich and Bongaerts, Johannes and Siegert, Petra}, title = {Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans}, series = {FEBS Open Bio}, volume = {13}, journal = {FEBS Open Bio}, number = {12}, publisher = {Wiley}, address = {Hoboken, NJ}, issn = {2211-5463}, doi = {10.1002/2211-5463.13723}, pages = {2224 -- 2238}, year = {2023}, abstract = {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.}, language = {en} } @article{FalkenbergKohnBottetal.2023, author = {Falkenberg, Fabian and Kohn, Sophie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822ᵀ}, series = {FEBS Open Bio}, volume = {13}, journal = {FEBS Open Bio}, number = {11}, publisher = {Wiley}, address = {Hoboken, NJ}, issn = {2211-5463}, doi = {10.1002/2211-5463.13701}, pages = {2035 -- 2046}, year = {2023}, abstract = {Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5\% (w/v) SDS and 5\% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.}, language = {en} } @techreport{SiegertBongaertsWagneretal.2022, author = {Siegert, Petra and Bongaerts, Johannes and Wagner, Torsten and Sch{\"o}ning, Michael Josef and Selmer, Thorsten}, title = {Abschlussbericht zum Projekt zur {\"U}berwachung biotechnologischer Prozesse mittels Diacetyl-/Acetoin-Biosensor und Evaluierung von Acetoin-Reduktasen zur Verwendung in Biotransformationen}, address = {Aachen}, organization = {FH Aachen}, pages = {16 Seiten}, year = {2022}, language = {de} } @article{JanusAchtsnichtTempeletal.2023, author = {Janus, Kevin Alexander and Achtsnicht, Stefan and Tempel, Laura and Drinic, Aleksaner and Kopp, Alexander and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Influence of fibroin membrane composition and curing parameters on the performance of a biodegradable enzymatic biosensor manufactured from Silicon-Free Carbon}, series = {Physica status solidi : pss. A, Applications and materials science}, volume = {220}, journal = {Physica status solidi : pss. A, Applications and materials science}, number = {22}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6300 (Print)}, doi = {10.1002/pssa.202300081}, pages = {Artikel 2300081}, year = {2023}, abstract = {Herein, fibroin, polylactide (PLA), and carbon are investigated for their suitability as biocompatible and biodegradable materials for amperometric biosensors. For this purpose, screen-printed carbon electrodes on the biodegradable substrates fibroin and PLA are modified with a glucose oxidase membrane and then encapsulated with the biocompatible material Ecoflex. The influence of different curing parameters of the carbon electrodes on the resulting biosensor characteristics is studied. The morphology of the electrodes is investigated by scanning electron microscopy, and the biosensor performance is examined by amperometric measurements of glucose (0.5-10 mM) in phosphate buffer solution, pH 7.4, at an applied potential of 1.2 V versus a Ag/AgCl reference electrode. Instead of Ecoflex, fibroin, PLA, and wound adhesive are tested as alternative encapsulation compounds: a series of swelling tests with different fibroin compositions, PLA, and Ecoflex has been performed before characterizing the most promising candidates by chronoamperometry. Therefore, the carbon electrodes are completely covered with the particular encapsulation material. Chronoamperometric measurements with H2O2 concentrations between 0.5 and 10 mM enable studying the leakage current behavior.}, language = {en} } @article{FalkenbergVossBottetal.2023, author = {Falkenberg, Fabian and Voß, Leonie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {New robust subtilisins from halotolerant and halophilic Bacillaceae}, series = {Applied Microbiology and Biotechnology}, volume = {107}, journal = {Applied Microbiology and Biotechnology}, publisher = {Springer Nature}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12553-w}, pages = {3939 -- 3954}, year = {2023}, abstract = {The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5\% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.}, language = {en} } @article{HaegerWirgesTanzmannetal.2023, author = {Haeger, Gerrit and Wirges, Jessika and Tanzmann, Nicole and Oyen, Sven and Jolmes, Tristan and Jaeger, Karl-Erich and Sch{\"o}rken, Ulrich and Bongaerts, Johannes and Siegert, Petra}, title = {Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis}, series = {Microbial Cell Factories}, journal = {Microbial Cell Factories}, number = {22}, publisher = {Springer Nature}, issn = {1475-2859}, doi = {10.1186/s12934-023-02079-1}, pages = {Article number: 77 (2023)}, year = {2023}, abstract = {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.}, language = {en} } @article{MolinnusJanusFangetal.2022, author = {Molinnus, Denise and Janus, Kevin Alexander and Fang, Anyelina C. and Drinic, Aleksander and Achtsnicht, Stefan and K{\"o}pf, Marius and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Thick-film carbon electrode deposited onto a biodegradable fibroin substrate for biosensing applications}, series = {Physica status solidi (a)}, volume = {219}, journal = {Physica status solidi (a)}, number = {23}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6319}, doi = {10.1002/pssa.202200100}, pages = {1 -- 9}, year = {2022}, abstract = {This study addresses a proof-of-concept experiment with a biocompatible screen-printed carbon electrode deposited onto a biocompatible and biodegradable substrate, which is made of fibroin, a protein derived from silk of the Bombyx mori silkworm. To demonstrate the sensor performance, the carbon electrode is functionalized as a glucose biosensor with the enzyme glucose oxidase and encapsulated with a silicone rubber to ensure biocompatibility of the contact wires. The carbon electrode is fabricated by means of thick-film technology including a curing step to solidify the carbon paste. The influence of the curing temperature and curing time on the electrode morphology is analyzed via scanning electron microscopy. The electrochemical characterization of the glucose biosensor is performed by amperometric/voltammetric measurements of different glucose concentrations in phosphate buffer. Herein, systematic studies at applied potentials from 500 to 1200 mV to the carbon working electrode (vs the Ag/AgCl reference electrode) allow to determine the optimal working potential. Additionally, the influence of the curing parameters on the glucose sensitivity is examined over a time period of up to 361 days. The sensor shows a negligible cross-sensitivity toward ascorbic acid, noradrenaline, and adrenaline. The developed biocompatible biosensor is highly promising for future in vivo and epidermal applications.}, language = {en} }