Kerstin Weil

Publications of Kerstin Weil

• Selective (R)-3-hydroxylation of FA by Stenotrophomonas maltophilia.

  Authors: Kerstin Weil, Patrick Gruber, Frank Heckel, Dag Harmsen, Peter Schreier

  Lipids. 04/2002; 37(3):317-23.

  Soil samples were screened for microorganisms selectively transforming FA. One of the isolated strains was identified as the bacterium Stenotrophomonas maltophilia by its phenotypic features andSoil samples were screened for microorganisms selectively transforming FA. One of the isolated strains was identified as the bacterium Stenotrophomonas maltophilia by its phenotypic features and genotypic characterization by sequencing the ribosomal RNA gene. Using linoleic acid as substrate resulted in the formation of two major compounds. After liquid chromatographic isolation and separation, their structures were elucidated by HPLC-tandem MS, GC-MS, and NMR techniques to be 3-hydroxy-Z6-dodecenoic acid and 3-hydroxy-Z5,Z8-tetradecadienoic acid. In additional experiments, other FA, such as a-linolenic, oleic, palmitoleic, myristoleic, and cis-vaccenic acids, were converted to 3-hydroxylated metabolites of shorter chain lengths as well. Determination of the enantiomeric composition revealed highly enriched (R)-hydroxylation (88-98% enantiomeric excess).

• Absolute configuration of 3-hydroxy acids formed by Stenotrophomonas maltophilia: application of multidimensional gas chromatography and circular dichroism spectroscopy.

  Authors: Kerstin Weil, Hans-Ulrich Humpf, Wilfried Schwab, Peter Schreier

  Chirality. 02/2002; 14(1):51-8.

  The soil bacterium Stenotrophomonas maltophilia was found to transform various long-chain fatty acids selectively into 3-hydroxy fatty acids of shorter chain length. Their chiral evaluation wasThe soil bacterium Stenotrophomonas maltophilia was found to transform various long-chain fatty acids selectively into 3-hydroxy fatty acids of shorter chain length. Their chiral evaluation was performed by multidimensional gas chromatography (MDGC) on modified cyclodextrin phase comparing the enantiodistribution of 1,3-diol formed without loss of stereochemical information from a representative microbial product with those of synthetic (3RS)- and (3S)-1,3-diols. Enantiomeric excesses of 84-98% (R) were determined for the microbially produced 3-hydroxy acids. In addition, the CD exciton chirality method was applied to determine their absolute configuration. Derivatization with 9-anthryldiazomethane and 2-naphthoylimidazole led to the required bichromophoric structures. Their CD spectra displayed a positive first Cotton effect around 254 nm and a negative second Cotton effect around 237 nm, which confirmed the (R)-configuration of the bacterial products.

• 3-(R)-Hydroxysäuren als Produkte selektiven Fettsäureabbaus [Elektronische Ressource] : Studien zur [beta]-Oxidation in Stenotrophomonas maltophilia /

  Authors: Kerstin. Weil

  Würzburg, Univ., Diss., 2001. Computerdatei im Fernzugriff.

• 3-(R)-Hydroxysäuren als Produkte selektiven Fettsäureabbaus

  Authors: Kerstin Weil

  In der vorliegenden Arbeit werden Studien zur selektiven bakteriellen Hydroxylierung von Fettsäuren vorgestellt. Unter Verwendung von Linolsäure als Substrat wurden aus Bodenproben verschiedeneIn der vorliegenden Arbeit werden Studien zur selektiven bakteriellen Hydroxylierung von Fettsäuren vorgestellt. Unter Verwendung von Linolsäure als Substrat wurden aus Bodenproben verschiedene Mikroorganismen isoliert, die polare Metabolite bildeten. Die phänotypische und genotypische Charakterisierung eines Stammes führte zu dessen Identifizierung als Stenotrophomonas maltophilia. Die Strukturaufklärung der drei Hauptreaktionsprodukte erfolgte mittels Hochleistungsflüssigchromatographie-Massenspektrometrie (HPLC-MS), Gaschromatographie-Massenspektrometrie (GC-MS) sowie ein- und zweidimensionalen NMR-Experimenten (1H-NMR, 13C-NMR, 13C-DEPT, H/H-COSY, HMQC, HMBC). Linolsäure wurde von Stenotrophomonas maltophilia zu 3-Hydroxy-Z6-dodecensäure, 3-Hydroxy-Z5,Z8-tetradecadiensäure und 3-Hydroxy-Z7,Z10-hexadecadiensäure umgesetzt. In einem anschließenden Substratscreening wurden 32 Verbindungen als Edukte für die Biotransformation eingesetzt und so die strukturellen Voraussetzungen ermittelt, die für eine effiziente Umsetzung von Fettsäuren durch Stenotrophomonas maltophilia notwendig sind. Zum Einsatz kamen Substrate mit unterschiedlicher Anzahl an C-Atomen sowie mit Variationen bezüglich Anzahl, Position und Konformation von Doppelbindungen. Weiterhin wurden Substanzen verwendet, die bereits funktionelle Gruppen im Molekül aufwiesen (z. B. Ricinolsäure). Die Bestimmung der Enantiomerenverteilung der bakteriell gebildeten 3-Hydroxysäuren mittels multidimensionaler Gaschromatographie (MDGC) ergab einen deutlichen Enantiomerenüberschuss (ee 84 – 98 Prozent). Die Aufklärung der Absolutkonfiguration erfolgte über die Synthese von Dodecan-1,3-diolen und deren anschließende Analytik mittels MDGC. Zusätzlich wurde die Konfiguration mit Hilfe der CD Exciton Chirality-Methode bestimmt. Weiterhin wurde untersucht, ob die bakteriell gebildeten 3-Hydroxysäuren als Substrate oder Inhibitoren des Enzyms Lipoxygenase L-1 aus Sojabohnen fungieren. Die im Rahmen dieser Arbeit durchgeführten Studien zur Darstellung von optisch aktiven 3-Hydroxysäuren belegen das Potential des Bodenbakteriums Stenotrophomonas maltophilia, exogen zugeführte Fettsäuren im Rahmen der b-Oxidation zu kettenverkürzten, an Position 3 hydroxylierten Metaboliten abzubauen. Dabei liegen jedoch deutliche Abweichungen zur b-Oxidation in anderen Organismen vor, die auf Unterschieden in der Enzymausstattung bzw. deren Aktivität beruhen. Durch die gewonnenen Erkenntnisse zum b-Oxidationsmechanismus in Stenotrophomonas maltophilia kann diese Aktivität durch geeignete Substratauswahl gezielt zur Synthese von optisch aktiven 3-Hydroxysäuren eingesetzt werden, deren chemische Synthese gegenüber dieser Biotransformation deutlich schwieriger zu realisieren ist. Für solche Verbindungen besteht in der organischen Synthese von Naturstoffen wie Pheromonen, Vitaminen und Antibiotika Bedarf. The available work presents studies on the selective bacterial hydroxylation of fatty acids. In a screening procedure using linoleic acid as substrate different microorganisms were isolated from soil samples and tested for their ability to form polar products. The phenotypic and genotypic characterisation of one of these strains led to its identification as Stenotrophomonas maltophilia. Structure elucidation of the three major reaction products was carried out by high performance liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry as well as one and two-dimensional NMR experiments (1H-NMR, 13C-NMR, 13C-DEPT, H/H COSY, HMQC, HMBC). Linoleic acid was converted by Stenotrophomonas maltophilia 3-hydroxy-Z6-dodecenoic acid, 3-hydroxy-Z5,Z8-tetradecadienoic acid and 3-hydroxy-Z7,Z10-hexadecadienoic acid. In a following screening 32 compounds were used as substrates for the biotransformation to determine the structural prerequisites, which are necessary for an efficient conversion of fatty acids by Stenotrophomonas maltophilia. On the basis of the results obtained with linoleic acid further fatty acids with 18 carbon atoms differing in number, position and configuration of available double bonds were used. Further compounds with differing chain length as well as substrates already containing functional groups were employed. Determination of the enantiomeric composition of the bacterially formed 3-hydroxy-acids by multidimensional gas chromatography (MDGC) resulted in a clear dominance of one of the enantiomers (ee 84 - 98 Prozent). Assigning the absolute configuration was carried out by syntheses of dodecane-1,3-diols and their analysis by MDGC. In addition, the CD exciton chirality method was applied to determine the absolute configuration of eight biotransformation products with different structural properties such as number and position of available double bonds. Kinetic studies using lipoxygenase L-1 from soy beans showed that the bacterial products neither act as substrates nor as inhibitors of this enzyme. The studies regarding the synthesis of optically active 3-hydroxy acids, executed in the context of this work, demonstrated the potential of the soil bacterium Stenotrophomonas maltophilia, to degrade exogenously supplied fatty acids to chain-shortened hydroxylated metabolites by b-oxidation. Clear deviations to the b-oxidation in other organisms are present, which are based on differences in the enzyme equipment or their activity. Based on the achieved results concerning the mechanism of b-oxidation in Stenotrophomonas maltophilia this activity can be used for the synthesis of optically active 3-hydroxy acids whose chemical synthesis is more difficult in relation to this biotransformation. For such compounds requirement exists in the organic synthesis of natural substances such as pheromones, vitamins and antibiotics.