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 - PAT A1 - Siegert, Petra A1 - Mussmann, Nina A1 - O'Connell, Timothy A1 - Maurer, Karl-Heinz T1 - Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift] T1 - Novel proteases and means containing said proteases [Internationale Patentanmeldung] Y1 - 2010 SP - 1 EP - 30 PB - Deutsches Patent- und Markenamt / WIPO CY - München / Genf ER - TY - PAT A1 - Siegert, Petra A1 - Baumstark, Rebecca A1 - Kluin, Cornelia A1 - O'Connell, Timothy A1 - Maurer, Karl-Heinz A1 - Hellmuth, Hendrik T1 - Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift] Y1 - 2010 SP - 1 EP - 30 PB - Deutsches Patent- und Markenamt CY - München ER - TY - PAT A1 - Siegert, Petra A1 - Spitz, Astrid A1 - Maurer, Karl-Heinz T1 - Neue Proteasen und Mittel enthaltend diese Proteasen [Offenlegungsschrift] T1 - Novel proteases and compositions comprising these proteases [Internationale Patentanmeldung] Y1 - 2010 SP - 1 EP - 31 PB - Deutsches Patentamt / WIPO CY - München / Genf ER - TY - PAT A1 - Siegert, Petra A1 - Spitz, Astrid A1 - Maurer, Karl-Heinz T1 - Wasch- und Reinigungsmittel enthaltend Proteasen aus Bacillus pumilus [Offenlegungsschrift] T1 - Detergents and cleaning agents containing proteases from bacillus pumilus [Europäische Patentschrift / Internationale Patentanmeldung] Y1 - 2010 SP - 1 EP - 20 PB - Deutsches Patentamt / Europäisches Patentamt / WIPO CY - München / Den Hague / Genf ER - TY - PAT A1 - O'Connell, Timothy A1 - Siegert, Petra A1 - Evers, Stefan A1 - Bongaerts, Johannes A1 - Weber, Thomas A1 - Maurer, Karl-Heinz A1 - Bessler, Cornelius T1 - Wasch- oder Reinigungsmittel mit gesteigerter Waschkraft [Offenlegungsschrift] T1 - Method from improving the cleaning action of a detergent of cleaning agent [US Patentanmeldung] Y1 - 2010 SP - 1 EP - 34 PB - Deutsches Patentamt CY - München ER - TY - PAT A1 - O'Connell, Timothy A1 - Siegert, Petra A1 - Maurer, Karl-Heinz A1 - Schiedel, Marc-Steffen A1 - Vockenroth, Inga Kerstin T1 - Method for improving the cleaning action of a detergent or cleaning agent [Internationale Patentanmeldung] T1 - Verfahren zur Verbesserung der Reinigungsleistung eines Wasch- oder Reinigungsmittels Y1 - 2010 SP - 1 EP - 15 PB - WIPO CY - Genf ER - TY - JOUR A1 - Degering, Christian A1 - Eggert, Thorsten A1 - Puls, Michael A1 - Bongaerts, Johannes A1 - Evers, Stefan A1 - Maurer, Karl-Heinz A1 - Jaeger, Karl-Erich T1 - Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and herologous signal peptides JF - Applied and environmental microbiology N2 - 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. Y1 - 2010 U6 - http://dx.doi.org/10.1128/AEM.01146-10 SN - 1098-5336 (E-Journal); 0003-6919 (Print); 0099-2240 (Print) VL - 76 IS - 19 SP - 6370 EP - 6378 PB - American Society for Microbiology CY - Washington, DC ER -