@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} } @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{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} } @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{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{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} } @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} } @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{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} } @article{IdingSiegertMeschetal.1998, author = {Iding, Hans and Siegert, Petra and Mesch, K. and Pohl, Martina}, title = {Application of α-keto acid decarboxylases in biotransformations}, series = {Biochimica et biophysica acta (BBA) - Protein structure and molecular enzymology}, volume = {Vol. 1385}, journal = {Biochimica et biophysica acta (BBA) - Protein structure and molecular enzymology}, number = {Iss. 2}, issn = {1879-2588 (E-Journal); 0167-4838 (Print)}, pages = {307 -- 322}, year = {1998}, language = {en} }