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

structure elucidation techniques such as the direct couplings of HPLC withPDA-MS and -MS/MS or by means of MALDI-TOF-MS and -TOF-TOF-MS, which enable high throughput screening for metabolite libraries. Thesetasks in combination with biological tests are an essential part of the entirebiocombinatorial process for rapid screening of modified natural products.Our results of the investigation of thousands of extracts from fungi andbacteria strains by using a combination of on-line and off-linechromatographic and spectroscopic methods showed that more than 30% ofthe identified metabolites are novel compounds.This talk presents as examples only the rapid identification of novel biologicactive cyclic depsideptides from Fusarium and Xylariaceae strains as well asfrom Pseudomonas bacteria associated with pathogenic Phytophthoraspecies by means of MALDI-TOF-TOF, LC-ESI-Q-TOF-MS and -MS/MSas well as -H/D-Exchange-MS, -MS/MS and -Pseudo-MS 3 .NTP016Studies on Chalara fraxinea infection process of ashplants - Direct and rapid detection of the pathogenChalara fraxinea in plant tissue by means of massspectrometric techniquesT.L.H. Pham* 1 , I. Zaspel* 21 Institute for Ecology, University of Technology, Berlin, Germany2 Federal Research Institute for Rural Areas, Forestry and Fisheries (vTI,Institute of Forest Genetics, Waldsieversdorf, GermanySince several years, Chalara fraxinea has caused a large-scale decline of ash(Fraxinus excelsior) in Europe. The disease affects trees of various ages innatural as well as in artificial environments. Infected trees die rapidlyindependent of their age-class, irrespective of site conditions andregeneration methods. Therefore, EPPO had assigned this new invasivepathogen into its Alert List of potential threats.Our tests (with different modes of artificial inoculation on 2-year-old ashseedlings) revealed that inoculation with conidia elicits the colonizing of thephloem of the whole shoot by the pathogen within a few weeks resulting inwilting of leaves, drying of buds of canopy, and culminating in death ofplant. The course of disease was much stronger in the sample inoculatedafter flushing, whereas inoculation before flushing resulted in a slow diseaseoutbreak with weak symptoms.Investigation of cell extracts of C. fraxinea grown on MEA, CMA, and PDAby means of LC-MS or MALDI-MS showed a range of unknown secondarymetabolites, especially the series of [M+Na] + at m/z 1133.7, 1175.7, and1217.7 Da. The same metabolites were detected in the tissues of dead plantsin nature or after artificial inoculation with the pathogen too, partly in highconcentration. A transport of secondary metabolites of C. fraxinea or of thepathogen itself from the inoculation spot to plant roots was observed. Thehighest concentration was found near stem basis, root collar, and primaryroot whereas these metabolites were not found in segments aboveinoculation spot. No metabolites of C. fraxinea were detected in those plantswhich did not sicken after treatment with conidia of C. fraxinea, e.g. becauseinoculation occurred before flushing.For the first time, the pathogen C. fraxinea is directly and rapidly detectedin-vitro in tissues of diseased ash by means of MS techniques. Thus, theseMS based high-throughput-screening methods can very effectively completeor replace the time consuming and expensive microbiological isolationprocedures for detection of the pathogen C. fraxinea and can be used torapidly test ash genotypes for resistance / susceptibility to C. fraxineainfection, respectively. They could also be verified in natural regeneration ofan ash stand infected by the pathogen in the natural environment.NTP017Identification of Microorganisms of Veterinary Origin byMALDI-TOF Mass SpectrometryA. Rothkamp 1 , A.A. Hassan 1 , C. Lämmler 2 , M. Hijazin 2 , T. Eisenberg 3 ,M. Zschöck 3 , M. Timke 4 , M. Kostrzewa* 41 GD - Animal Health Service Deventer, Deventer, Netherlands2 Institute of Pharmacology and Toxicology, Justus-Liebig-University,Gießen, Germany3 Hessian State Office Laboratory, Gießen, Germany4 Bruker Daltonik GmbH, Bremen, Germanyconsumables and laboratory staff has to be highly qualified. Faster, morereliable, automated and cost effective methods for identification anddifferentiation of microorganisms of veterinary origins will become moreimportant. In this study we evaluated the MALDI Biotyper system (BrukerDaltonik) for identification and differentiation of 197 different isolatesobtained from veterinary routine diagnostic (n = 125) and reference strains(n = 72). 121 field isolates were previously identified with morphologicaland biochemical tests, e.g. API test system. The bacteria used in this studyincluded isolates of genera Actinobaculum, Actinobacillus, Avibacterium,Bordetella, Brachyspira, Campylobacter, Clostridium, Corynebacterium,Cronobacter, Enterobacter, Enterococcus, Erysipelothrix, Gallibacterium,Histophilus, Janthinobacterium, Kluyvera, Mannheimia, Micrococcus,Moraxella, Nocardia, Ornithobacterium, Pantoea, Pasteurella, Proteus,Salmonella, Staphylococcus, Streptococcus and Yersinia. A total of 169(85.8 %) isolates were identified to species level, 14 (7.1 %) to genus leveland 14 (7.1 %) isolates were not reliably identified. For further improvementof the system bacterial strains from the genera Avibacterium, Brachyspira,Riemerella, Staphylococcus, Streptococcus, and Taylorella will be added tothe database. The present results show that MALDI-TOF MS is a fast andreliable automated method for identification of most species of veterinaryorigin.NTP018Modification of Extremozymes by Non-canonical AminoAcidsN. Winkelmann* 1 , L. Merkel 2 , B. Klippel 1 , N. Budisa 2 , G. Antranikian 11 Technical Microbiology, University of Technology, Harburg, Germany2 Department of Biocatalysis, Technical University, Berlin, GermanySynthetic Biology offers a broad spectrum of techniques for de novo designor re-design of enzymes. In this context, genetic code engineering allows theresidue-specific replacement of a particular canonical amino acid(s) at allpositions in the protein sequence, with non-canonical one(s) without needfor DNA mutagenesis [1]. Non-canonical (mainly synthetic) amino acidsexhibit distinct features from canonical ones and usually endow substituted(i.e. congeneric) proteins with novel structural and functional features [2].This concept was applied on the well characterized enzymes fromextremophilic microorganisms such as lipase, amylase and cellulase. Theyare interesting candidates for these modifications, since they show catalyticactivity at extremes of pH and temperature and tolerate high solventconcentrations. The lipase from the extreme thermophileThermoanaerobacter thermohydrosulfuricus has been modified and thegenerated lipase congeners showed enhanced activation and significantshifts of optimal temperature and pH [4].In this study, we introduce two synthetic enzymes from thermophilicmicroorganisms by expanding their amino acid repertoire. An α-amylasefrom the archaeon Pyrococcus woesei [5] and a cellulase from an anaerobicthermophile have been cloned and heterologously expressed in strains ofEscherichia coli. Translation was reprogrammed by introducing differentnon-canonical amino acid analogs of methionine, proline, phenylalanine andtryptophan to the cultures. A complete substitution of methionine residuesby L-norleucin, proline residues by (4-fluoro)proline and phenylalanineresidues by D,L-(4-fluorophenyl)alanine was observed. The specificreactions of the obtained enzyme congeners were compared with the wildtypeproteins and their substrate spectra. The contribution of syntheticmodifications to enzyme activity, stability and efficiency will be presentedand discussed.[1] Lepthien, S. et al (2010): In Vivo Double and Triple Labeling of Proteins Using Synthetic AminoAcids. Angew. Chem. Int. Ed. 49 (32), 5446-5450; Angew. Chem. 122, 5576-5581.[2] Merkel, L. et al (2010): Parallel Incorporation of Different Fluorinated Amino Acids: On the Wayto Teflon Proteins. ChemBioChem. 11 (11), 1505-1507.[3] Budisa, N. et al (2010): Residue-specific global fluorination of Candida Antarctica lipase B inPichia pastoris. Mol. Biosyst. 6 (9), 1630-1639.[4] Hoesl, M. G. et al (2010): Lipase congeners designed by genetic code engineering.ChemCatChem, published online: 19 Oct. 2010 (DOI: 10.1002/cctc.201000253).[5] Linden, A. et al (2000): Single-step purification of a recombinant thermostable a-amylase aftersolubilization of the enzyme from insoluble aggregates, Journal of Chromatography, 737: 253-259.Identification of microorganisms of veterinary origin is usually done byusing traditional culture depending and biochemical methods or by semiautomated methods. These methods are time consuming, need a lot ofspektrum | Tagungsband 2011