TY - JOUR A1 - Ribitsch, D. A1 - Karl, W. A1 - Birner-Gruenberger, R. A1 - Gruber, K. A1 - Eiteljoerg, I. A1 - Remler, P. A1 - Wieland, S. A1 - Siegert, Petra A1 - Maurer, Karl-Heinz A1 - Schwab, H. T1 - C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli JF - Journal of biotechnology N2 - Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease. Y1 - 2010 U6 - http://dx.doi.org/10.1016/j.jbiotec.2010.09.947 SN - 1873-4863 (E-Journal); 0168-1656 (Print) VL - 150 IS - 3 SP - 408 EP - 416 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Ribitsch, D. A1 - Heumann, S. A1 - Karl, W. A1 - Gerlach, J. A1 - Leber, R. A1 - Birner-Gruenberger, R. A1 - Gruber, K. A1 - Eiteljoerg, I. A1 - Remler, P. A1 - Siegert, Petra A1 - Lange, J. A1 - Maurer, Karl-Heinz A1 - Berg, G. A1 - Guebitz, G. M. A1 - Schwab, H. T1 - Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli JF - Journal of biotechnology N2 - A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2. KW - Alginate beads KW - Stenotrophomonas maltophilia KW - Detergent protease Y1 - 2012 U6 - http://dx.doi.org/10.1016/j.jbiotec.2011.09.025 SN - 1873-4863 (E-Journal); 0168-1656 (Print) VL - 157 IS - 1 SP - 140 EP - 147 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Reisert, Steffen A1 - Schneider, Benno A1 - Geissler, Hanno A1 - Gompel, Matthias van A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Multi-sensor chip for the investigation of different types of metal oxides for the detection of H2O2 in the ppm range JF - physica status solidi (a) N2 - In this work, a multi-sensor chip for the investigation of the sensing properties of different types of metal oxides towards hydrogen peroxide in the ppm range is presented. The fabrication process and physical characterization of the multi-sensor chip are described. Pure SnO2 and WO3 as well as Pd- and Pt-doped SnO2 films are characterized in terms of their sensitivity to H2O2. The sensing films have been prepared by drop-coating of water-dispensed nano-powders. A physical characterization, including scanning electron microscopy and X-ray diffraction analysis of the deposited metal-oxide films, was done. From the measurements in hydrogen peroxide atmosphere, it could be shown, that all of the tested metal oxide films are suitable for the detection of H2O2 in the ppm range. The highest sensitivity and reproducibility was achieved using Pt-doped SnO2. Calibration plot of a SnO2, WO3, Pt-, and Pd-doped SnO2 gas sensor for H2O2 concentrations in the ppm range. Y1 - 2013 SN - 1862-6319 VL - 210 IS - 5 SP - 898 EP - 904 PB - Wiley CY - Weinheim ER - TY - JOUR A1 - Reisert, Steffen A1 - Henkel, Hartmut A1 - Schneider, Andreas A1 - Schäfer, Daniel A1 - Friedrich, Peter A1 - Berger, Jörg A1 - Schöning, Michael Josef T1 - Development of a handheld sensor system for the online measurement of hydrogen peroxide in aseptic filling systems JF - Physica Status Solidi (A). 207 (2010), H. 4 Y1 - 2010 SN - 1862-6300 N1 - Special Issue: Engineering of Functional Interfaces EnFI 2009 SP - 913 EP - 918 ER - TY - JOUR A1 - Reisert, Steffen A1 - Henkel, H. A1 - Schneider, A. A1 - Schäfer, D. A1 - Friedrich, P. A1 - Berger, J. A1 - Schöning, Michael Josef T1 - Entwicklung eines Handheld-Sensorsystems für die „On-line“-Messung der H2O2-Konzentration in aseptischen Entkeimungsprozessen JF - 9. Dresdner Sensor-Symposium : Dresden, 07.-09. Dezember 2009 / Gerlach, Gerald ; Hauptmann, Peter [Hrsg.] Y1 - 2009 SN - 978-3-941298-44-6 N1 - Dresdner Sensor-Symposium ; (9, 2009, Dresden) SP - 285 EP - 288 PB - TUDpress CY - Dresden ER - TY - JOUR A1 - Reisert, Steffen A1 - Geissler, Hanno A1 - Flörke, Rudolf A1 - Weiler, Christian A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Characterisation of aseptic sterilisation processes using an electronic nose JF - International journal of nanotechnology Y1 - 2013 SN - 1475-7435 (Print) 7141-8151 (Online) VL - Vol. 10 IS - No. 5-7 SP - 470 EP - 484 PB - Inderscience Enterprises CY - Genève ER - TY - JOUR A1 - Reisert, Steffen A1 - Geissler, Hanno A1 - Flörke, Rudolf A1 - Näther, Niko A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Towards a multi-sensor system for the evaluation of aseptic processes employing hydrogen peroxide vapour (H2O2) JF - Physica status solidi (a) : applications and material science. 208 (2011), H. 6 Y1 - 2011 SN - 1862-6319 SP - 1351 EP - 1356 PB - Wiley CY - Weinheim ER - TY - JOUR A1 - Reisert, Steffen A1 - Geissler, H. A1 - Weiler, C. A1 - Wagner, P. A1 - Schöning, Michael Josef T1 - Multiple sensor-type system for monitoring the microbicidal effectiveness of aseptic sterilisation processes JF - Food control N2 - The present work describes a novel multiple sensor-type system for the real-time analysis of aseptic sterilisation processes employing gaseous hydrogen peroxide (H2O2) as a sterilant. The inactivation kinetics of Bacillus atrophaeus by gaseous H2O2 have been investigated by means of a methodical calibration experiment, taking into account the process variables H2O2 concentration, humidity and gas temperature. It has been found that the microbicidal effectiveness at H2O2 concentrations above 2% v/v is largely determined by the concentration itself, while at lower H2O2 concentrations, the gas temperature and humidity play a leading role. Furthermore, the responses of different types of gas sensors towards the influencing factors of the sterilisation process have been analysed within the same experiment. Based on a correlation established between the inactivation kinetics and the sensor responses, a calorimetric H2O2 sensor and a metal-oxide semiconductor (MOX) sensor have been identified as possible candidates for monitoring the microbicidal effectiveness of aseptic sterilisation processes employing gaseous H2O2. Therefore, two linear models that describe the relationship between sensor response and microbicidal effectiveness have been proposed. Y1 - 2015 U6 - http://dx.doi.org/10.1016/j.foodcont.2014.07.063 SN - 1873-7129 (E-Journal); 0956-7135 (Print) VL - 47 SP - 615 EP - 622 ER - TY - JOUR A1 - Reisert, Steffen A1 - Geissler, H. A1 - Florke, R. A1 - Wagner, P. A1 - Wagner, Torsten A1 - Schöning, Michael Josef T1 - Controlling aseptic sterilization processes by means of a multi-sensor system Y1 - 2011 N1 - 2011 IEEE Workshop on Merging Fields of Computational Intelligence and Sensor Technology ; 11.-15. April 2011 Paris, France SP - 18 EP - 22 PB - IEEE CY - New York ER - TY - JOUR A1 - Rachinger, Michael A1 - Bauch, Melanie A1 - Strittmatter, Axel A1 - Bongaerts, Johannes A1 - Evers, Stefan A1 - Maurer, Karl-Heinz A1 - Daniel, Rolf A1 - Liebl, Wolfgang A1 - Liesegang, Heiko A1 - Ehrenreich, Armin T1 - Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis JF - Journal of biotechnology Y1 - 2013 SN - 1873-4863 (E-Journal); 0168-1656 (Print) VL - Vol. 164 IS - Iss. 4 SP - 365 EP - 369 PB - Elsevier CY - Amsterdam ER -