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Koch, Linda ; Wunderlich, F. Thomas ; Seibler, Jost ; Könner, A. Christine ; Hampel, Brigitte ; Irlenbusch, Sigrid ; Brabant, Georg ; Kahn, C. Ronald ; Schwenk, Frieder ; Brüning, Jens C.
Handtke, Stefan ; Volland, Sonja ; Methling, Karen ; Albrecht, Dirk ; Becher, Dörte ; Nehls, Jenny ; Bongaerts, Johannes ; Maurer, Karl-Heinz ; Lalk, Michael ; Liesegang, Heiko ; Voigt, Birgit ; Daniel, Rolf ; Hecker, Michael
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC–MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.
Wendorff, Marion ; Eggert, Thorsten ; Pohl, Martina ; Dresen, Carola ; Müller, Michael ; Jaeger, Karl-Erich ; Sprenger, Georg A. ; Schürmann, Melanie ; Schürmann, Martin ; Johnen, Sandra ; Sprenger, Gerda ; Sahm, Hermann ; Inoue, Tomoyuki ; Schörken, Ulrich ; Breittaupt, Holger ; Frölich, Bettina ; Heim, Petra ; Iding, Hans ; Juchem, Bettina ; Siegert, Petra ; Kula, Maria-Regina ; Weckbecker, Andrea ; Hummel, Werner ; Fessner, Wolf-Dieter ; Elling, Lothar ; Wolberg, Michael ; Bode, Silke ; Feldmann, Ralf ; Geilenkirchen, Petra ; Schubert, Thomas ; Walter, Lydia ; Dünnwald, Thomas ; Demir, Ayhan S. ; Kolter-Jung, Doris ; Nitsche, Adam ; Dünkelmann, Pascal ; Cosp, Annabel ; Lingen, Bettina
C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Ribitsch, Doris ; Karl, Wolfgang ; Birner-Gruenberger, R. ; Gruber, K. ; Eiteljoerg, I. ; Remler, Peter ; Wieland, S. ; Siegert, Petra ; Maurer, Karl-Heinz ; Schwab, H.
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.
Siegert, Petra ; Iding, Hans ; Baumann, Martin ; McLeish, Michael J. ; Kenyon, George L. ; Pohl, Martina
Al-Kaidy, Huschyar ; Duwe, Anna ; Huster, Manuel ; Muffler, Kai ; Schlegel, Christin ; Tim, Sieker ; Stadtmüller, Ralf ; Tippkötter, Nils ; Ulber, Roland
For several thousand years, biotechnology and its associated technical processes have had a great impact on the development of mankind. Based on empirical methods, in particular for the production of foodstuffs and daily commodities, these disciplines have become one of the most innovative future issues. Due to the increasing detailed understanding of cellular processes, production strains can now be optimized. In combination with modern bioprocesses, a variety of bulk and fine chemicals as well as pharmaceuticals can be produced efficiently. In this article, some of the current trends in biotechnology are discussed.
The terms bioeconomy and biorefineries are used for a variety of processes and developments. This short introduction is intended to provide a delimitation and clarification of the terminology as well as a classification of current biorefinery concepts. The basic process diagrams of the most important biorefinery types are shown.