@article{RibitschHeumannKarletal.2012, author = {Ribitsch, D. and Heumann, S. and Karl, W. and Gerlach, J. and Leber, R. and Birner-Gruenberger, R. and Gruber, K. and Eiteljoerg, I. and Remler, P. and Siegert, Petra and Lange, J. and Maurer, Karl-Heinz and Berg, G. and Guebitz, G. M. and Schwab, H.}, title = {Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli}, series = {Journal of biotechnology}, volume = {157}, journal = {Journal of biotechnology}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2011.09.025}, pages = {140 -- 147}, year = {2012}, abstract = {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.}, language = {en} } @incollection{SiegertPohlKneenetal.2004, author = {Siegert, Petra and Pohl, Martina and Kneen, Malea M. and Pogozheva, Irina D. and Kenyon, George L. and McLeish, Michael J.}, title = {Exploring the substrate specificity of benzoylformate decarboxylase, pyruvate decarboxylase, and benzaldehyde lyase}, series = {Thiamine : catalytic mechanisms in normal and disease states / ed. by Frank Jordan ...}, booktitle = {Thiamine : catalytic mechanisms in normal and disease states / ed. by Frank Jordan ...}, publisher = {Dekker}, address = {New York, NY}, isbn = {0-8247-4062-9}, pages = {275 -- 290}, year = {2004}, language = {en} } @article{SiegertMcLeishBaumannetal.2005, author = {Siegert, Petra and McLeish, Michael J. and Baumann, Martin and Iding, Hans and Kneen, Malea M. and Kenyon, George L. and Pohl, Martina}, title = {Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida}, series = {Protein engineering, design, and selection : peds}, volume = {Vol. 18}, journal = {Protein engineering, design, and selection : peds}, number = {Iss. 7}, issn = {1460-213X (E-Journal); 1741-0134 (E-Journal); 0269-2139 (Print); 1741-0126 (Print)}, pages = {345 -- 357}, year = {2005}, language = {en} } @article{NiehausGaborWielandetal.2011, author = {Niehaus, F. and Gabor, E. and Wieland, S. and Siegert, Petra and Maurer, Karl-Heinz and Eck, J.}, title = {Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases}, series = {Microbial biotechnology}, volume = {Vol. 4}, journal = {Microbial biotechnology}, number = {Iss. 6}, publisher = {Springer}, address = {Berlin}, issn = {1432-0614 (E-Journal); 0171-1741 (Print); 0175-7598 (Print); 0340-2118 (Print)}, pages = {767 -- 776}, year = {2011}, language = {en} } @article{DuennwaldDemirSiegertetal.2000, author = {D{\"u}nnwald, Thomas and Demir, Ayhan S. and Siegert, Petra and Pohl, Martina and M{\"u}ller, Michael}, title = {Enantioselective Synthesis of (S)-2-Hydroxypropanone Derivatives by Benzoylformate Decarboxylase Catalyzed C-C Bond Formation}, series = {European journal of organic chemistry}, volume = {Vol. 2000}, journal = {European journal of organic chemistry}, number = {Iss. 11}, issn = {0365-5490 (E-Journal); 1099-0690 (E-Journal); 0075-4617 (Print); 0170-2041 (Print); 0947-3440 (Print); 1434-193X (Print); 1434-243X (Print)}, pages = {2161 -- 2170}, year = {2000}, language = {en} } @article{DuenkelmannKolterJungNitscheetal.2002, author = {D{\"u}nkelmann, Pascal and Kolter-Jung, Doris and Nitsche, Adam and Demir, Ayhan S. and Siegert, Petra and Lingen, Bettina and Baumann, Martin and Pohl, Martina and M{\"u}ller, Michael}, title = {Development of a donor-acceptor concept for enzymatic cross-coupling reactions of adehydes : the first asymmetric cross-benzoin condensation}, series = {Journal of the American Chemical Society}, volume = {Vol. 124}, journal = {Journal of the American Chemical Society}, issn = {1520-5126 (E-Journal); 0002-7863 (Print)}, pages = {12084 -- 12085}, year = {2002}, language = {en} } @article{MolinnusMuschallikGonzalezetal.2018, author = {Molinnus, Denise and Muschallik, Lukas and Gonzalez, Laura Osorio and Bongaerts, Johannes and Wagner, Torsten and Selmer, Thorsten and Siegert, Petra and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Development and characterization of a field-effect biosensor for the detection of acetoin}, series = {Biosensors and Bioelectronics}, volume = {115}, journal = {Biosensors and Bioelectronics}, publisher = {Elsevier}, address = {Amsterdam}, doi = {10.1016/j.bios.2018.05.023}, pages = {1 -- 6}, year = {2018}, abstract = {A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO₂/Ta₂O₅/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.}, language = {en} } @article{MolinnusBaeckerSiegertetal.2015, author = {Molinnus, Denise and B{\"a}cker, Matthias and Siegert, Petra and Willenberg, H. and Poghossian, Arshak and Keusgen, M. and Sch{\"o}ning, Michael Josef}, title = {Detection of Adrenaline Based on Substrate Recycling Amplification}, series = {Procedia Engineering}, volume = {120}, journal = {Procedia Engineering}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1877-7058}, doi = {10.1016/j.proeng.2015.08.708}, pages = {540 -- 543}, year = {2015}, abstract = {An amperometric enzyme biosensor has been applied for the detection of adrenaline. The adrenaline biosensor has been prepared by modification of an oxygen electrode with the enzyme laccase that operates at a broad pH range between pH 3.5 to pH 8. The enzyme molecules were immobilized via cross-linking with glutaraldehyde. The sensitivity of the developed adrenaline biosensor in different pH buffer solutions has been studied.}, language = {en} } @article{WeldenSeverinsPoghossianetal.2022, author = {Welden, Melanie and Severins, Robin and Poghossian, Arshak and Wege, Christina and Bongaerts, Johannes and Siegert, Petra and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Detection of acetoin and diacetyl by a tobacco mosaic virus-assisted field-effect biosensor}, series = {Chemosensors}, volume = {10}, journal = {Chemosensors}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2227-9040}, doi = {10.3390/chemosensors10060218}, pages = {Artikel 218}, year = {2022}, abstract = {Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte-insulator-semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716ᵀ is immobilized via biotin-streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage-current, capacitance-voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.}, language = {en} } @article{DuennwaldDemirSiegertetal.2001, author = {D{\"u}nnwald, Thomas and Demir, Ayhan S. and Siegert, Petra and Pohl, Martina and M{\"u}ller, Michael}, title = {ChemInform Abstract: Enantioselective synthesis of (S)-2-Hydroxypropanone derivatives by Benzoylformate Decarboxylase Catalyzed C—C Bond Formation}, series = {Cheminform}, volume = {Vol. 32}, journal = {Cheminform}, number = {Iss. 4}, issn = {1522-2667 (E-Journal); 0931-7597 (Print)}, pages = {Publ. online}, year = {2001}, language = {en} } @incollection{WendorffEggertPohletal.2007, author = {Wendorff, Marion and Eggert, Thorsten and Pohl, Martina and Dresen, Carola and M{\"u}ller, Michael and Jaeger, Karl-Erich and Sprenger, Georg A. and Sch{\"u}rmann, Melanie and Sch{\"u}rmann, Martin and Johnen, Sandra and Sprenger, Gerda and Sahm, Hermann and Inoue, Tomoyuki and Sch{\"o}rken, Ulrich and Breittaupt, Holger and Fr{\"o}lich, Bettina and Heim, Petra and Iding, Hans and Juchem, Bettina and Siegert, Petra and Kula, Maria-Regina and Weckbecker, Andrea and Hummel, Werner and Fessner, Wolf-Dieter and Elling, Lothar and Wolberg, Michael and Bode, Silke and Feldmann, Ralf and Geilenkirchen, Petra and Schubert, Thomas and Walter, Lydia and D{\"u}nnwald, Thomas and Demir, Ayhan S. and Kolter-Jung, Doris and Nitsche, Adam and D{\"u}nkelmann, Pascal and Cosp, Annabel and Lingen, Bettina}, title = {Catalytic asymmetric synthesis : section 2.2}, series = {Asymmetric synthesis with chemical and biological methods / ed. by Dieter Enders ...}, booktitle = {Asymmetric synthesis with chemical and biological methods / ed. by Dieter Enders ...}, publisher = {Wiley-VCH}, address = {Weinheim}, isbn = {978-3-527-31473-7}, pages = {298 -- 413}, year = {2007}, language = {en} } @article{JablonskiMuenstermannNorketal.2021, author = {Jablonski, Melanie and M{\"u}nstermann, Felix and Nork, Jasmina and Molinnus, Denise and Muschallik, Lukas and Bongaerts, Johannes and Wagner, Torsten and Keusgen, Michael and Siegert, Petra and Sch{\"o}ning, Michael Josef}, title = {Capacitive field-effect biosensor applied for the detection of acetoin in alcoholic beverages and fermentation broths}, series = {physica status solidi (a) applications and materials science}, volume = {218}, journal = {physica status solidi (a) applications and materials science}, number = {13}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6319}, doi = {10.1002/pssa.202000765}, pages = {7 Seiten}, year = {2021}, abstract = {An acetoin biosensor based on a capacitive electrolyte-insulator-semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance-voltage, and constant capacitance methods, respectively.}, language = {en} } @article{RibitschKarlBirnerGruenbergeretal.2010, author = {Ribitsch, D. and Karl, W. and Birner-Gruenberger, R. and Gruber, K. and Eiteljoerg, I. and Remler, P. and Wieland, S. and Siegert, Petra and Maurer, Karl-Heinz and Schwab, H.}, title = {C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli}, series = {Journal of biotechnology}, volume = {150}, journal = {Journal of biotechnology}, number = {3}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2010.09.947}, pages = {408 -- 416}, year = {2010}, abstract = {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.}, language = {en} } @inproceedings{SiegertIdingBaumannetal.2000, author = {Siegert, Petra and Iding, Hans and Baumann, Martin and McLeish, Michael J. and Kenyon, George L. and Pohl, Martina}, title = {Broadening of the substrate spectra of two ThDP-dependent decarboxylases using site-directed-mutagenesis}, series = {Proceedings of the 4th International Congress on Biochemical Engineering : 17 and 18 February 2000, Stuttgart}, booktitle = {Proceedings of the 4th International Congress on Biochemical Engineering : 17 and 18 February 2000, Stuttgart}, organization = {International Congress on Biochemical Engineering <4, 2000, Stuttgart>}, isbn = {3-8167-5570-4}, pages = {38 -- 42}, year = {2000}, language = {en} } @article{FalkenbergRahbaFischeretal.2022, author = {Falkenberg, Fabian and Rahba, Jade and Fischer, David and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T}, series = {FEBS Open Bio}, volume = {12}, journal = {FEBS Open Bio}, number = {10}, publisher = {Wiley}, address = {Hoboken, NJ}, issn = {2211-5463}, doi = {10.1002/2211-5463.13457}, pages = {1729 -- 1746}, year = {2022}, abstract = {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.}, 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} } @misc{BankowskiHoffmannWadleetal.2003, author = {Bankowski, Bernhard and Hoffmann, Daniele and Wadle, Armin and Siegert, Petra and S{\"a}ttler, Andrea and Gerke, Thomas}, title = {Beta-Glucuronidase-Inhibitoren in Deodorantien und Antitranspirantien [Offenlegungsschrift]}, publisher = {Deutsches Patent- und Markenamt / Europ{\"a}isches Patentamt / WIPO}, address = {M{\"u}nchen / Den Hague / Genf}, pages = {1 -- 24}, year = {2003}, language = {de} } @article{IdingDuennwaldGreineretal.2000, author = {Iding, Hans and D{\"u}nnwald, Thomas and Greiner, Lasse and Liese, Andreas and M{\"u}ller, Michael and Siegert, Petra and Gr{\"o}tzinger, Joachim and Demir, Ayhan S. and Pohl, Martina}, title = {Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones}, series = {Chemistry - a European journal}, volume = {Vol. 6}, journal = {Chemistry - a European journal}, number = {Iss. 8}, issn = {1521-3765 (E-Journal); 0947-6539 (Print)}, pages = {1483 -- 1495}, year = {2000}, language = {en} } @misc{BanowskiWadleSiegert2004, author = {Banowski, Bernhard and Wadle, Armin and Siegert, Petra}, title = {Arylsulfatase-Inhibitoren in Deodorantien und Antitranspirantien [Offenlegungsschrift]}, publisher = {Deutsches Patent- und Markenamt / Europ{\"a}isches Patentamt}, address = {M{\"u}nchen / Den Hague}, pages = {1 -- 15}, year = {2004}, language = {de} } @misc{BanowskiWadleSiegert2003, author = {Banowski, Bernhard and Wadle, Armin and Siegert, Petra}, title = {Arylsulfatase-Inhibitoren in Deodorantien und Antitranspirantien [Offenlegungsschrift]}, publisher = {Deutsches Patent- und Markenamt}, address = {M{\"u}nchen}, pages = {1 -- 20}, year = {2003}, language = {de} }