TY - JOUR A1 - Falkenberg, Fabian A1 - Kohn, Sophie A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822ᵀ JF - FEBS Open Bio N2 - 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. KW - Bacillaceae KW - Biotechnological application KW - Broad pH spectrum KW - Subtilases KW - Subtilisin Y1 - 2023 U6 - http://dx.doi.org/10.1002/2211-5463.13701 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 11 SP - 2035 EP - 2046 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Morais, Paulo V. A1 - Suman, Pedro H. A1 - Schöning, Michael Josef A1 - Siqueira Junior, José R. A1 - Orlandi, Marcelo O. T1 - Layer-by-layer film based on Sn₃O₄ nanobelts as sensing units to detect heavy metals using a capacitive field-effect sensor platform JF - Chemosensors N2 - Lead and nickel, as heavy metals, are still used in industrial processes, and are classified as “environmental health hazards” due to their toxicity and polluting potential. The detection of heavy metals can prevent environmental pollution at toxic levels that are critical to human health. In this sense, the electrolyte–insulator–semiconductor (EIS) field-effect sensor is an attractive sensing platform concerning the fabrication of reusable and robust sensors to detect such substances. This study is aimed to fabricate a sensing unit on an EIS device based on Sn₃O₄ nanobelts embedded in a polyelectrolyte matrix of polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA) using the layer-by-layer (LbL) technique. The EIS-Sn₃O₄ sensor exhibited enhanced electrochemical performance for detecting Pb²⁺ and Ni²⁺ ions, revealing a higher affinity for Pb²⁺ ions, with sensitivities of ca. 25.8 mV/decade and 2.4 mV/decade, respectively. Such results indicate that Sn₃O₄ nanobelts can contemplate a feasible proof-of-concept capacitive field-effect sensor for heavy metal detection, envisaging other future studies focusing on environmental monitoring. KW - Sn₃O₄ KW - nanobelts KW - field-effect sensor KW - LbL films KW - heavy metals Y1 - 2023 U6 - http://dx.doi.org/10.3390/chemosensors11080436 SN - 2227-9040 N1 - This article belongs to the Special Issue The Application of Electrochemical Sensors or Biosensors Based on Nanomaterials VL - 11 IS - 8 PB - MDPI CY - Basel ER - TY - JOUR A1 - Janus, Kevin Alexander A1 - Achtsnicht, Stefan A1 - Drinic, Aleksander A1 - Kopp, Alexander A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Transient magnesium-based thin-film temperature sensor on a flexible, bioabsorbable substrate for future medical applications JF - Applied Research N2 - 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. KW - Silk fibroin KW - Polylactide acid KW - Bioabsorbable KW - Resistive temperature detector Y1 - 2023 U6 - http://dx.doi.org/10.1002/appl.202300102 SN - 2702-4288 (Print) N1 - Corresponding author: Michael Josef Schöning IS - Accepted manuscript PB - Wiley-VCH ER - TY - JOUR A1 - Karschuck, Tobias A1 - Schmidt, Stefan A1 - Achtsnicht, Stefan A1 - Poghossian, Arshak A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Multiplexing system for automated characterization of a capacitive field-effect sensor array JF - Physica Status Solidi A N2 - 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. KW - Capacitive field-effect sensor KW - Gold nanoparticles KW - Label-free detection KW - Multicell KW - Multiplexing Y1 - 2023 U6 - http://dx.doi.org/10.1002/pssa.202300265 SN - 1862-6300 (Print) SN - 1862-6319 (Online) N1 - Corresponding author: Michael Josef Schöning VL - 220 IS - 22 PB - Wiley-VCH CY - Weinheim 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 - Falkenberg, Fabian A1 - Rahba, Jade A1 - Fischer, David A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T JF - FEBS Open Bio N2 - Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future. KW - Alkalihalobacillus okhensis KW - detergent protease KW - halotolerant protease KW - high-alkaline subtilisin KW - oxidative stable protease Y1 - 2022 U6 - http://dx.doi.org/10.1002/2211-5463.13457 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 12 IS - 10 SP - 1729 EP - 1746 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Pourshahidi, Ali Mohammad A1 - Engelmann, Ulrich M. A1 - Offenhäusser, Andreas A1 - Krause, Hans-Joachim T1 - Resolving ambiguities in core size determination of magnetic nanoparticles from magnetic frequency mixing data JF - Journal of Magnetism and Magnetic Materials N2 - Frequency mixing magnetic detection (FMMD) has been widely utilized as a measurement technique in magnetic immunoassays. It can also be used for the characterization and distinction (also known as “colourization”) of different types of magnetic nanoparticles (MNPs) based on their core sizes. In a previous work, it was shown that the large particles contribute most of the FMMD signal. This leads to ambiguities in core size determination from fitting since the contribution of the small-sized particles is almost undetectable among the strong responses from the large ones. In this work, we report on how this ambiguity can be overcome by modelling the signal intensity using the Langevin model in thermodynamic equilibrium including a lognormal core size distribution fL(dc,d0,σ) fitted to experimentally measured FMMD data of immobilized MNPs. For each given median diameter d0, an ambiguous amount of best-fitting pairs of parameters distribution width σ and number of particles Np with R2 > 0.99 are extracted. By determining the samples’ total iron mass, mFe, with inductively coupled plasma optical emission spectrometry (ICP-OES), we are then able to identify the one specific best-fitting pair (σ, Np) one uniquely. With this additional externally measured parameter, we resolved the ambiguity in core size distribution and determined the parameters (d0, σ, Np) directly from FMMD measurements, allowing precise MNPs sample characterization. Y1 - 2022 U6 - http://dx.doi.org/10.1016/j.jmmm.2022.169969 SN - 0304-8853 VL - 563 IS - In progress, Art. No. 169969 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Engelmann, Ulrich M. A1 - Pourshahidi, Mohammad Ali A1 - Shalaby, Ahmed A1 - Krause, Hans-Joachim T1 - Probing particle size dependency of frequency mixing magnetic detection with dynamic relaxation simulation JF - Journal of Magnetism and Magnetic Materials N2 - Biomedical applications of magnetic nanoparticles (MNP) fundamentally rely on the particles’ magnetic relaxation as a response to an alternating magnetic field. The magnetic relaxation complexly depends on the interplay of MNP magnetic and physical properties with the applied field parameters. It is commonly accepted that particle core size is a major contributor to signal generation in all the above applications, however, most MNP samples comprise broad distribution spanning nm and more. Therefore, precise knowledge of the exact contribution of individual core sizes to signal generation is desired for optimal MNP design generally for each application. Specifically, we present a magnetic relaxation simulation-driven analysis of experimental frequency mixing magnetic detection (FMMD) for biosensing to quantify the contributions of individual core size fractions towards signal generation. Applying our method to two different experimental MNP systems, we found the most dominant contributions from approx. 20 nm sized particles in the two independent MNP systems. Additional comparison between freely suspended and immobilized MNP also reveals insight in the MNP microstructure, allowing to use FMMD for MNP characterization, as well as to further fine-tune its applicability in biosensing. Y1 - 2022 U6 - http://dx.doi.org/10.1016/j.jmmm.2022.169965 SN - 0304-8853 VL - 563 IS - In progress, Art. No. 169965 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Falkenberg, Fabian A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae JF - Frontiers in Microbiology N2 - The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates. Y1 - 2022 U6 - http://dx.doi.org/10.3389/fmicb.2022.1017978 SN - 1664-302X VL - 2022 IS - 13 PB - Frontiers CY - Lausanne 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 -