@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}
}
@misc{SiegertMussmannO'Connelletal.2010,
  author    = {Siegert, Petra and Mussmann, Nina and O'Connell, Timothy and Maurer, Karl-Heinz},
  title     = {Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift]},
  publisher = {Deutsches Patent- und Markenamt / WIPO},
  address   = {M{\"u}nchen / Genf},
  pages     = {1 -- 30},
  year      = {2010},
  language  = {de}
}
@misc{SiegertBaumstarkKluinetal.2010,
  author    = {Siegert, Petra and Baumstark, Rebecca and Kluin, Cornelia and O'Connell, Timothy and Maurer, Karl-Heinz and Hellmuth, Hendrik},
  title     = {Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift]},
  publisher = {Deutsches Patent- und Markenamt},
  address   = {M{\"u}nchen},
  pages     = {1 -- 30},
  year      = {2010},
  language  = {de}
}
@misc{SiegertSpitzMaurer2010,
  author    = {Siegert, Petra and Spitz, Astrid and Maurer, Karl-Heinz},
  title     = {Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift]},
  publisher = {Deutsches Patentamt / WIPO},
  address   = {M{\"u}nchen / Genf},
  pages     = {1 -- 31},
  year      = {2010},
  language  = {de}
}
@misc{SiegertSpitzMaurer2010,
  author    = {Siegert, Petra and Spitz, Astrid and Maurer, Karl-Heinz},
  title     = {Wasch- und Reinigungsmittel enthaltend Proteasen aus Bacillus pumilus [Offenlegungsschrift]},
  publisher = {Deutsches Patentamt / Europ{\"a}isches Patentamt / WIPO},
  address   = {M{\"u}nchen / Den Hague / Genf},
  pages     = {1 -- 20},
  year      = {2010},
  language  = {de}
}
@misc{O'ConnellSiegertEversetal.2010,
  author    = {O'Connell, Timothy and Siegert, Petra and Evers, Stefan and Bongaerts, Johannes and Weber, Thomas and Maurer, Karl-Heinz and Bessler, Cornelius},
  title     = {Wasch- oder Reinigungsmittel mit gesteigerter Waschkraft [Offenlegungsschrift]},
  publisher = {Deutsches Patentamt},
  address   = {M{\"u}nchen},
  pages     = {1 -- 34},
  year      = {2010},
  language  = {de}
}
@misc{O'ConnellSiegertMaureretal.2010,
  author    = {O'Connell, Timothy and Siegert, Petra and Maurer, Karl-Heinz and Schiedel, Marc-Steffen and Vockenroth, Inga Kerstin},
  title     = {Method for improving the cleaning action of a detergent or cleaning agent [Internationale Patentanmeldung]},
  publisher = {WIPO},
  address   = {Genf},
  pages     = {1 -- 15},
  year      = {2010},
  language  = {en}
}
@article{DegeringEggertPulsetal.2010,
  author    = {Degering, Christian and Eggert, Thorsten and Puls, Michael and Bongaerts, Johannes and Evers, Stefan and Maurer, Karl-Heinz and Jaeger, Karl-Erich},
  title     = {Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and herologous signal peptides},
  series = {Applied and environmental microbiology},
  volume    = {76},
  journal   = {Applied and environmental microbiology},
  number    = {19},
  publisher = {American Society for Microbiology},
  address   = {Washington, DC},
  issn      = {1098-5336 (E-Journal); 0003-6919 (Print); 0099-2240 (Print)},
  doi       = {10.1128/AEM.01146-10},
  pages     = {6370 -- 6378},
  year      = {2010},
  abstract  = {Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.},
  language  = {en}
}