VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

enzyme was purified via metal ion affinity chromatography. Additionally,imidazole and excess NaCl were removed from the concentrated proteinfraction by using PD-10 desalting columns (GE Healthcare), because of theirdisturbing effects for the measurement of enzyme concentration and activity.The enzymatic assay was performed anaerobically, using a photometeradjusted to 37 °C. The pH was kept at 9.2 with a KHCO 3 buffer, and thereaction was started with the 1,3-PD samples. The overproduced 1,3-PDdehydrogenase was characterized concerning its stability, substratespecificity, and the optimal pH and temperature for its activity. Furthermore,a calibration curve between 0 and 60 mM 1,3-PD with a correlationcoefficient of 0.992 could be obtained for the overproduced enzyme, whichallows for the determination of 1,3-PD concentrations in culture samples ofclostridial strains grown on glycerol as substrate.GWP008Effective biocatalytic synthesis of D-Rhamnose, a majorbuilding block of carbohydrate-based vaccinesM. Pitz*, S. Dorscheid, F. GiffhornInstitute for Microbiology, Saarland University, Saarbrücken, GermanyD-Rhamnose (6-deoxy-D-mannose) is rare in nature but it is the majorconstituent of immunogenic oligosaccharides (O-antigens) of various humanand plant pathogenic gram-negative bacteria. Therefore, the availability ofD-Rhamnose is of concern to syntheses of carbohydrate-based vaccinesagainst human pathogens [1]. As chemical synthesis of D-Rhamnose and itsextraction from bacterial lipopolysaccharides are both tedious and lowyielding,we have developed a concise biocatalytic route to D-Rhamnosewith yields >40%.The route started from 6-deoxy-D-glucose 1 which was quantitativelyconverted in water to 6-deoxy-D-glucosone 2 (30 g · l -1· h -1 ) using acatalytically improved pyranose 2-oxidase variant [2]. Downstreamprocessing of 2 was facile and comprised ultrafiltration and lyophilisation(>90%). Solid 2 was dissolved in deionized water and quantitatively reduced(9 g · l -1· h -1 ) to a mixture of D-Rhamnose 3 (42%) and 6-deoxy-D-glucose 1(58%) with 1,5-Anhydro-D-fructose-Reductase (AFR) [3] and cosubstrateregeneration. When the conversion was complete, residual D-glucose fromcosubstrate regeneration and 6-deoxy-D-glucose 1 were oxidized to thecorresponding gluconic acids with glucose oxidase. Downstream processingof D-Rhamnose 3 comprised ultrafiltration, removal of charged compoundsby ion-exchange chromatography and lyophilisation to give solid 3.[1] Fauré, R. et al. (2007) Org. Biomol. Chem. 5: 2704-8.[2] Dorscheid, S. (2009) PhD thesis, Saarland University.[3] Kühn, A. et al. (2006) Appl. Environ. Micobiol. 72: 1248-57.GWP009L-Sorbitol-Dehydrogenase (LSDH) from Bradyrhizobiumjaponicum USDA110: Cloning and Characterisation of anInteresting Enzyme for Rare Sugar SynthesisS. Gauer* 1 , H. Otten 2 , L. Lo Leggio 2 , F. Giffhorn 1 , G.-W. Kohring 11 Insitute for Microbiology, Saarland University, Saarbruecken, Germany2 Biophysical Chemistry, University of Copenhagen, Copenhagen, DenmarkThe rare sugar D-sorbose is an interesting synthon for pharmaceuticalapplications and can be produced from easily prepared L-sorbitol by LSDH[1]. BLAST search with the N-terminal amino acid sequence of theStenotrophomonas maltophilia enzyme [1] listed putative enzymes with bestsimilarities for an assumed ribitol-DH of Bradyrhizobium japonicum. Thegene was amplified, tagged with histidines and heterologously expressed.The enzyme was biochemically characterized collecting data on temperatureand pH-optimum, isoelectric point, substrate spectrum and subunitcomposition. First structural data suggest temperature stability andcrystallisation experiments are in progress. The enzyme exhibited highactivity for D-sorbitol transformation to D-fructose but also reasonableactivity with L-sorbitol resulting in D-sorbose as the single product. Thereaction products were analysed via HPLC, the cofactor is regenerated withlactate-dehydrogenase. A cost effective co-factor regeneration system forthese procedures can be achieved with electrochemical methods as has beenshown for DSDH from Rhodobacter sphaeroides [2].[1] Brechtel, E. et al (2002): Appl Environ Microbiol. 68(2), 582-587.[2] Gajdzik, J. et al (2007): J. Solid State Electrochem. 11, 144-149.GWP010Development of genetic tools aiming at strainimprovement in Bacillus pumilusS. Wemhoff* 1 , J. Bongaerts 2 , S. Evers 2 , K.-H. Maurer 2 , F. Meinhardt 11 Insitute for Molecular Microbiology and Biotechnology, WestphalianWilhelms-University, Münster, Germany2 Henkel AG & Co. KGaA, Biotechnology, Düsseldorf, GermanyMembers of the Gram positive endospore forming genus Bacillus areintensively used for the industrial production of secreted enzymes such asproteases, amylases, and chitinases. Recently, the only sparsely investigatedspecies Bacillus pumilus got into focus due to its high secretion capacity forextracellular enzymes serving as an alternative producer strain for industrialenzyme production. However, the scientific knowledge concerning B.pumilus is currently rather poor. Thus, genetic tools have to be developedand applied for strain improvement of B. pumilus. Here, we focus on thedevelopment, improvement, and application of basic genetic tools forBacillus pumilus such as transformation techniques for plasmid-transfer(PEG-mediated protoplast transformation, electroporation, conjugation,natural competence), procedures for gene replacement and direct knock outs(induced competence with pMMcomK [1] , upp counter selection system [2] ),generation of stable and safety strains (spoIV, uvrBA and recA deletionmutants), establishment of random mutagenesis systems (mariner-Himar1transposon system for Bacilli [ 3] ) or construction of reporter gene systems.This work is supported by the Bundesministerium für Bildung undForschung (BMBF, grant no. 0315594C).[1] Hoffmann K. et al (2010): Appl. Environm. Microbiol., Vol. 76 (15), p. 5046-5057.[2] Fabret C. et al. (2002): Mol. Microbiol., Vol. 46 (1), p. 25-36.[3] Le Breton Y. et al (2006): Appl. Environm. Microbiol., Vol. 72 (1), p. 327-333.GWP011Characterisation of Friulimicin Production duringCultivation of Actinoplanes friuliensis in a bioreactorA. Steinkämper* 1 , N. Wagner 1 , A. Wolf 2 , R. Masuch 2 , J. Hofmann 1,2 ,D. Schwartz 1 , R. Biener 11 Faculty of Natural Sciences/Biotechnology, Universitiy of AppliedSciences, Esslingen, Germany2 micro-biolytics GmbH, Esslingen, GermanyFriulimicin is a lipopeptide antibiotic which is active against a broad rangeof multiresistant gram-positive bacteria such as methicillin-resistantEnterococcus spec. and Staphylococcus aureus (MRE, MRSA) strains. Theproducer strain of Friulimicin, Actinoplanes friuliensis, is a Gram positivesoil-inhabiting bacterium which belongs to the group of rare actinomycetes.A. friuliensis is a filamentous growing bacterium having a complex lifecycle, which includes morphological differentiation.For the characterization of Friulimicin biosynthesis, A. friuliensis wascultivated in a bioreactor under defined and controlled conditions. Achemically defined production medium, especially developed for A.friuliensis, was used. This defined medium is a prerequisite for thequantitative analysis of cell metabolism during the cultivations. A newdeveloped middle infrared spectroscopy method (AquaSpec Technology,micro-biolytics GmbH) was applied to analyse substrates and metabolites.In order to improve the understanding of the complex regulatory network ofthe friulimicin biosynthesis in A. friuliensis, a genome-scale network modelwill be developed and characterized. To validate the model and to performmetabolic flux analysis, data from the cultivations are integrated into thismodel. This model should give hints for directed genetically modificationsand development of process control strategies with the objective to redirectmetabolic fluxes towards Friulimicin production.GWP012In vitro characterization of Escherichia coli phage K1ERNA polymerase and its in vivo application for proteinproduction in Bacillus megateriumS. Stammen, F. Schuller, S. Dietrich, S. Wienecke, T. Knuuti, C. Finger,D. Jahn, R. Biedendieck*Institute of Microbiology, University of Technology, Braunschweig,GermanyGene „7” of Escherichia coli K1E phage, predicted to encode a DNAdependentRNA polymerase (RNAP), was cloned and heterologouslyspektrum | Tagungsband 2011