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Bioprozessentwicklung der AMDase katalysierten Synthese von Arylpropionsäure Derivaten
Citation Link: https://doi.org/10.15480/882.2013
Publikationstyp
Doctoral Thesis
Publikationsdatum
2019
Sprache
German
Author
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2018-11-28
Institut
Citation
Technische Universität Hamburg (2019)
Biocatalytic process design for the synthesis of enantiopure products is a promising and environmentally friendly alternative to conventional chemical synthesis routes and will increasingly be used in industry. The focus of this dissertation was the bioprocess development of an enzye catalyzed synthesis of arylpropionates, such as Ibuprofen or Naproxen.
The enantioselective step of synthesis is, for this purpose, performed by the Arylmalonate Decarboxylase (AMDase), which catalyzes the enantioselective decarboxylation of aromatic dicarbonic acids in a simple one-step reaction. The challenge here was to increase enzymatic stability under process conditions. Through immobilization onto a robust acrylic carrier, a 170-fold increase of stability was achieved, as well as an observed increase in the half-life of the free enzyme from 1.2 hours to a half-life of about 8.6 days of the immobilized enzyme with improved productivity. Additionally, the successful immobilization of the cell lysate led to the decrease in time and money spent on the process –as the enzyme purification step was not necessary anymore– and reveals a more stable enzyme preparation for the bioprocess.
The kinetic characterization of the immobilized enzyme in respect of naproxen malonic acid conversion reveals a KM of 22.1 ± 0.1 mM with an activity yield of about 40%. The slightly inhibiting effect of the product (Ki of 26.3 ± 1.4 mM) led to the selection of a batch mode for this bioprocess.
The preparative synthesis was realized in two stirred tank reactors. One, in a usual reactor where the immobilized enzyme is freely suspended in the reaction solution, and the other, in a rotating fixed bed reactor in which the enzyme was separated from the reaction mixture located in the stirrer element. In both stirred tank reactors, the synthesis of enantiopure (S)-naproxen (ee 99%) was realized in five repeated batch experiments with a productivity of 140 kg(S) Naproxen/kgAMDase-CLGIPL. After product isolation, by an easy acid induced product precipitation and subsequent product-drying, a yield of 92% of enantiopure (S)-naproxen was achieved.
The enantioselective step of synthesis is, for this purpose, performed by the Arylmalonate Decarboxylase (AMDase), which catalyzes the enantioselective decarboxylation of aromatic dicarbonic acids in a simple one-step reaction. The challenge here was to increase enzymatic stability under process conditions. Through immobilization onto a robust acrylic carrier, a 170-fold increase of stability was achieved, as well as an observed increase in the half-life of the free enzyme from 1.2 hours to a half-life of about 8.6 days of the immobilized enzyme with improved productivity. Additionally, the successful immobilization of the cell lysate led to the decrease in time and money spent on the process –as the enzyme purification step was not necessary anymore– and reveals a more stable enzyme preparation for the bioprocess.
The kinetic characterization of the immobilized enzyme in respect of naproxen malonic acid conversion reveals a KM of 22.1 ± 0.1 mM with an activity yield of about 40%. The slightly inhibiting effect of the product (Ki of 26.3 ± 1.4 mM) led to the selection of a batch mode for this bioprocess.
The preparative synthesis was realized in two stirred tank reactors. One, in a usual reactor where the immobilized enzyme is freely suspended in the reaction solution, and the other, in a rotating fixed bed reactor in which the enzyme was separated from the reaction mixture located in the stirrer element. In both stirred tank reactors, the synthesis of enantiopure (S)-naproxen (ee 99%) was realized in five repeated batch experiments with a productivity of 140 kg(S) Naproxen/kgAMDase-CLGIPL. After product isolation, by an easy acid induced product precipitation and subsequent product-drying, a yield of 92% of enantiopure (S)-naproxen was achieved.
Schlagworte
Biokatalyse
Immobilisiertes Enzym
Bioprozess
Kinetik
DDC Class
620: Ingenieurwissenschaften
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