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 - https://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 - 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 - CHAP A1 - Seibler, Jost A1 - Schwenk, Frieder T1 - Transgenic RNAi Applications in the Mouse T2 - Methods in Enzymology : Guide to Techniques in Mouse Development, Part B: Mouse Molecular Genetics. 2nd Edition Y1 - 2010 SN - 978-0-12-384880-2 N1 - Methods in Enzymology : Vol. 477 SP - 367 EP - 386 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Sieker, Tim A1 - Neuner, Andreas A1 - Dimitrova, Darina A1 - Tippkötter, Nils A1 - Bart, Hans-Jörg A1 - Heinzle, Elmar A1 - Ulber, Roland T1 - Grassilage als Rohstoff für die chemische Industrie JF - Chemie Ingenieur Technik N2 - Grassilage stellt einen nachwachsenden Rohstoff mit großem Potenzial dar. Neben Cellulose und Hemicellulose enthält sie auch organische Säuren, insbesondere Milchsäure. In einem Bioraffinerie-Projekt wird die Milchsäure aus der Silage isoliert und mit gentechnisch optimierten Stämmen zu L-Lysin weiterverarbeitet. Die Lignocellulose wird hydrolysiert und zu Ethanol fermentiert. Ein besonderes Augenmerk liegt auf der Integration der unterschiedlichen Prozesse sowie der einzelnen Prozessschritte zu einem Gesamtprozess, der sämtliche Inhaltsstoffe der Silage verwertet. Y1 - 2010 U6 - https://doi.org/10.1002/cite.201000088 SN - 1522-2640 VL - 82 IS - 8, Special Issue: Industrielle Nutzung nachwachsender Rohstoffe SP - 1153 EP - 1159 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Tippkötter, Nils A1 - Roikaew, Wipa A1 - Ulber, Roland A1 - Hoffmann, Alexander A1 - Denzler, Hans-Jörg A1 - Buchholz, Heinrich T1 - Paracoccus denitrificans for the effluent recycling during continuous denitrification of liquid food JF - Biotechnology Progress N2 - Nitrate is an undesirable component of several foods. A typical case of contamination with high nitrate contents is whey concentrate, containing nitrate in concentrations up to 25 l. The microbiological removal of nitrate by Paracoccus denitrificans under formation of harmless nitrogen in combination with a cell retention reactor is described here. Focus lies on the resource-conserving design of a microbal denitrification process. Two methods are compared. The application of polyvinyl alcohol-immobilized cells, which can be applied several times in whey feed, is compared with the implementation of a two step denitrification system. First, the whey concentrate's nitrate is removed by ion exchange and subsequently the eluent regenerated by microorganisms under their retention by crossflow filtration. Nitrite and nitrate concentrations were determined by reflectometric color measurement with a commercially available Reflectoquant® device. Correction factors for these media had to be determined. During the pilot development, bioreactors from 4 to 250 mg·L-1 and crossflow units with membrane areas from 0.02 to 0.80 m2 were examined. Based on the results of the pilot plants, a scaling for the exemplary process of denitrifying 1,000 tons per day is discussed. Y1 - 2010 U6 - https://doi.org/10.1002/btpr.384 SN - 8756-7938 VL - 26 IS - 3 SP - 756 EP - 762 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Ross, Jillian A1 - Plummer, Simon M. A1 - Rode, Anja A1 - Scheer, Nico A1 - Bower, Conrad C. A1 - Vogel, Ortwin A1 - Henderson, Colin J. A1 - Wolf, C. Roland A1 - Elcombe, Clifford R. T1 - Human constitutive androstane receptor (CAR) and pregnane X receptor (PXR) support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogens phenobarbital and chlordane in vivo JF - Toxicological Sciences N2 - Mouse nongenotoxic hepatocarcinogens phenobarbital (PB) and chlordane induce hepatomegaly characterized by hypertrophy and hyperplasia. Increased cell proliferation is implicated in the mechanism of tumor induction. The relevance of these tumors to human health is unclear. The xenoreceptors, constitutive androstane receptors (CARs), and pregnane X receptor (PXR) play key roles in these processes. Novel “humanized” and knockout models for both receptors were developed to investigate potential species differences in hepatomegaly. The effects of PB (80 mg/kg/4 days) and chlordane (10 mg/kg/4 days) were investigated in double humanized PXR and CAR (huPXR/huCAR), double knockout PXR and CAR (PXRKO/CARKO), and wild-type (WT) C57BL/6J mice. In WT mice, both compounds caused increased liver weight, hepatocellular hypertrophy, and cell proliferation. Both compounds caused alterations to a number of cell cycle genes consistent with induction of cell proliferation in WT mice. However, these gene expression changes did not occur in PXRKO/CARKO or huPXR/huCAR mice. Liver hypertrophy without hyperplasia was demonstrated in the huPXR/huCAR animals in response to both compounds. Induction of the CAR and PXR target genes, Cyp2b10 and Cyp3a11, was observed in both WT and huPXR/huCAR mouse lines following treatment with PB or chlordane. In the PXRKO/CARKO mice, neither liver growth nor induction of Cyp2b10 and Cyp3a11 was seen following PB or chlordane treatment, indicating that these effects are CAR/PXR dependent. These data suggest that the human receptors are able to support the chemically induced hypertrophic responses but not the hyperplastic (cell proliferation) responses. At this time, we cannot be certain that hCAR and hPXR when expressed in the mouse can function exactly as the genes do when they are expressed in human cells. However, all parameters investigated to date suggest that much of their functionality is maintained. Y1 - 2010 U6 - https://doi.org/10.1093/toxsci/kfq118 SN - 1096-0929 VL - 116 IS - 2 SP - 452 EP - 466 PB - Oxford University Press CY - Oxford ER -