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Discovery of novel Baeyer-Villiger monooxygenases and their application in organic synthesis.

  • The application of BVMOs in kinetic resolution is a versatile alternative for the synthesis of optically pure esters. Within this thesis BVMOs proved to be highly active against a broad range of linear and aryl aliphatic ketones yielding a variety of enantiopure products. Among the beta-hydroxy ketones several CHMOs and BVMOPsfl showed the best results (E > 100), whereas the application of the latter enzyme also allowed access to the abnormal esters (regioisomeric excess > 40%). Interestingly, some enzymes showed a reduced activity and selectivity with a growing chain length of the ketone, suggesting that middle-chain ketones (C8-C10) might be preferred. Moreover, the production of optically pure 1,2-diols was observed (yields 8-50%), resulting from an in vivo hydrolysis of the 2-hydroxy alkyl acetates. Regarding the N-protected beta-amino ketones, results were different. While the majority of CHMOs catalyzed linear substrates showing high enantioselectivities (for CHMOBrevi1 and CHMOBrachy E > 100, c = 40-50%), BVMOPsfl did not convert nitrogen bearing linear ketones, although this might also be justified with the methylcarbamate protecting group. Interestingly, the number of BVMOs catalyzing oxidation of spatially more demanding linear branched beta-amino ketones was greatly reduced, indicating steric hindrance that was also combined with a decrease in selectivity. Similar to the observation for beta-hydroxy ketones, also the 2 amino alkyl acetates hydrolyzed furnishing 2-amino alcohols (yields 9-52%). Moreover, hydrolysis of the “abnormal“ esters allowed an alternative access to valuable native and non-native β-amino acids. In a two step process, using CDMO from R. ruber and CAL-B, it was possible to generate N-protected (+)-beta-leucine. During kinetic resolutions of aryl aliphatic ketones it was observed that the highest enantio¬selectivities could be achieved utilizing HAPMOJD1, HAPMOACB and PAMO, enzymes typically preferring aromatic substrates. Biotransformation with 3-phenyl-2-butanone revealed an E-value > 100 for HAPMOJD1 (S-selective). Nevertheless, also BVMOPsfl converted this sub¬strate (E = 43), and also CHMOAcineto and CPMO oxidized it, although selectivity was rather low (E < 5). Interestingly, BVMOKT2440 was the only examined enzyme showing R selectivity (E = 13). Additionally, increasing the scale and performing biotransformation in a baffled flask could increase enantioselectivity of BVMOPsfl from E = 43 to 82. The discovery of novel enzymes with diverse properties is still a main goal of the biotechnological industry. Within these studies, two BVMOs (BVMOKT2440 and HAPMOJD1) could be successfully amplified from genomic DNA using different PCR-methods. Then, expression in E. coli was optimized, revealing that the reduction of expression temperature, implementation of E. coli JM109 or RosettaTM (DE3), possessing the pRARE plasmid to facilitate translation of rare codons in the latter case, and/or co-expression of chaperones (pGro7: GroEL/ES-familiy) could increase the amount of soluble and active protein. Both enzymes were subjected to biocatalysis and it was found that BVMOKT2440 preferentially oxidized linear ketones, while HAPMOJD1 dominantly converted aryl aliphatic ketones. The latter enzyme could be purified by anion exchange and affinity chromatography allowing examination of kinetic parameters. Thereby, HAPMOJD1 displayed lowest KM-values for acetophenone derivatives bearing their substituent in para-position (KM < 320 µM). Moreover, also aldehydes and heteroaromatic compounds were oxidized and also sulfoxidation was observed. Interestingly it was found, that both BVMO genes are located in the direct neighborhood of a dehydrogenase and a hydrolase. This led to the suggestion that these enzymes may be metabolically connected in the degradation of their natural substrate.
  • Innerhalb dieser Arbeit ist es gelungen mittels verschiedener PCR-Methoden eine Baeyer-Villiger Monooxygenase (BVMO) aus genomischer DNA von P. putida JD1 zu amplifiezieren. Das Gen wurde anschließend kloniert und in E. coli überexprimiert. Nach Expressionsoptimierung erfolgte die Aufreinigung (Anionenaustauscher und Affinitätschromatographie) und die Charakterisierung der neuen BVMO. Dabei zeigte sich, das besonders Acetophenon-Derivate, die den Substituenten in para-Position haben bevorzugt umgesetzt werden. Daneben wurden aber auch Aldehyde und Sulfide oxidiert. Das Temperaturmaximum lag bei 30°C, das pH-Maximum bei 8,5 (in TrisHCl-Puffer). Neben den molekularbiologischen Methoden wurde eine neue Substratklasse für BVMOs etabliert. Dafür wurden verschiedene beta-substituierte lineare, aliphatische Ketone chemisch synthetisiert und anschließend in Biotransformationen mit verschiedenen BVMOs in einer kinetischen Racematspaltung untersucht. Dabei zeigte sich, dass N-geschützte beta-Aminoketone, arylaliphatische Ketone und beta-Hydroxyketone sehr gute Substrate für BVMOs sind (E > 100, bei c = 50%). Außerdem konnte ein neuer enzymatischer Syntheseweg zur Darstellung von enantiomerenreinen 1,2-Diolen und optische aktiver Vorstufen von beta-Aminosäuren mit BVMOs aufgezeigt werden.

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Author: Jessica Rehdorf
Title Additional (German):Entdeckung neuer Baeyer-Villiger Monooxygenasen und deren Einsatzmöglichkeiten in der organischen Synthese.
Advisor:Prof. Uwe Bornscheuer
Document Type:Doctoral Thesis
Date of Publication (online):2010/01/26
Granting Institution:Ernst-Moritz-Arndt-Universität, Mathematisch-Naturwissenschaftliche Fakultät (bis 31.05.2018)
Date of final exam:2010/01/25
Release Date:2010/01/26
Tag:Baeyer-Villiger monooxygenases, regioselectivity
GND Keyword:Baeyer-Villiger-Oxidation, Biokatalyse, Monooxygenasen
Faculties:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie und Biochemie
DDC class:500 Naturwissenschaften und Mathematik / 540 Chemie