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 - Vahidpour, Farnoosh A1 - Guthman, Eric A1 - Arreola, Julia A1 - Alghazali, Yousef H. M. A1 - Wagner, Torsten A1 - Schöning, Michael Josef T1 - Assessment of Various Process Parameters for Optimized Sterilization Conditions Using a Multi-Sensing Platform JF - Foods N2 - In this study, an online multi-sensing platform was engineered to simultaneously evaluate various process parameters of food package sterilization using gaseous hydrogen peroxide (H₂O₂). The platform enabled the validation of critical aseptic parameters. In parallel, one series of microbiological count reduction tests was performed using highly resistant spores of B. atrophaeus DSM 675 to act as the reference method for sterility validation. By means of the multi-sensing platform together with microbiological tests, we examined sterilization process parameters to define the most effective conditions with regards to the highest spore kill rate necessary for aseptic packaging. As these parameters are mutually associated, a correlation between different factors was elaborated. The resulting correlation indicated the need for specific conditions regarding the applied H₂O₂ gas temperature, the gas flow and concentration, the relative humidity and the exposure time. Finally, the novel multi-sensing platform together with the mobile electronic readout setup allowed for the online and on-site monitoring of the sterilization process, selecting the best conditions for sterility and, at the same time, reducing the use of the time-consuming and costly microbiological tests that are currently used in the food package industry. KW - spore kill rate KW - sterility KW - aseptic parameters KW - multi-sensing platform KW - gaseous hydrogen peroxide Y1 - 2022 U6 - http://dx.doi.org/10.3390/foods11050660 SN - 2304-8158 N1 - This article belongs to the Special Issue "Sensors and Biosensors Application for Food Industries" VL - 11 IS - 5 PB - MDPI CY - Basel ER - TY - JOUR A1 - Vahidpour, Farnoosh A1 - Alghazali, Yousef A1 - Akca, Sevilay A1 - Hommes, Gregor A1 - Schöning, Michael Josef T1 - An Enzyme-Based Interdigitated Electrode-Type Biosensor for Detecting Low Concentrations of H₂O₂ Vapor/Aerosol JF - Chemosensors N2 - This work introduces a novel method for the detection of H₂O₂ vapor/aerosol of low concentrations, which is mainly applied in the sterilization of equipment in medical industry. Interdigitated electrode (IDE) structures have been fabricated by means of microfabrication techniques. A differential setup of IDEs was prepared, containing an active sensor element (active IDE) and a passive sensor element (passive IDE), where the former was immobilized with an enzymatic membrane of horseradish peroxidase that is selective towards H₂O₂. Changes in the IDEs’ capacitance values (active sensor element versus passive sensor element) under H₂O₂ vapor/aerosol atmosphere proved the detection in the concentration range up to 630 ppm with a fast response time (<60 s). The influence of relative humidity was also tested with regard to the sensor signal, showing no cross-sensitivity. The repeatability assessment of the IDE biosensors confirmed their stable capacitive signal in eight subsequent cycles of exposure to H₂O₂ vapor/aerosol. Room-temperature detection of H₂O₂ vapor/aerosol with such miniaturized biosensors will allow a future three-dimensional, flexible mapping of aseptic chambers and help to evaluate sterilization assurance in medical industry. Y1 - 2022 U6 - http://dx.doi.org/10.3390/chemosensors10060202 SN - 2227-9040 N1 - This article belongs to the Special Issue "Bioinspired Chemical Sensors and Micro-Nano Devices" VL - 10 IS - 6 PB - MDPI CY - Basel ER - TY - RPRT A1 - Siegert, Petra A1 - Bongaerts, Johannes A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Selmer, Thorsten T1 - Abschlussbericht zum Projekt zur Überwachung biotechnologischer Prozesse mittels Diacetyl-/Acetoin-Biosensor und Evaluierung von Acetoin-Reduktasen zur Verwendung in Biotransformationen Y1 - 2022 N1 - Laufzeit: 01.01.2016 – 31.12.2019 (verlängert bis 31.12.2020) Förderkennzeichen: 322-8.03.04.02-FH-Struktur 2016/02 Gefördert durch: Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen CY - Aachen 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 -