|Title:||Selective redox biocatalysis in multiphasic enzyme reactors|
|Abstract:||Reactions catalysed by oxidoreductases are indispensable for chemical synthesis. However, despite obvious advantages like mild reaction conditions and regio-, chemo-, and stereoselectivity, which simplifies product work-up, their implementation in the chemical industry is lacking. Therefore, an integrated approach involving catalyst and reaction engineering and proper reactor design is necessary for commercialising oxidoreductases for organic synthesis. Rational reaction design linking kinetic characterisation of the oxidoreductase and reaction optimisation formed the basis for this thesis. The overall aim of this PhD project was the application of 2-hydroxybiphenyl 3-monooxygenase (HbpA), for preparative scale synthesis of 3-phenylcatechol with an acceptable space time yield (STY). The key challenges limiting the biocatalytic process, namely biocatalyst operational stability, supply of the hydrophobic substrate, oxygen delivery, cofactor regeneration and mass transfer were identified and addressed. Formate dehydrogenase, FDH, was used for continuous NADH regeneration. Addition of the surfactant Tween 20 stabilised the enzymes in the presence of the organic solvent and allowed to recover 100% of the respective enzymatic activity opening the door for application of the biocatalysts in an aqueous/organic two-liquid phase segmented flow microreactor. A biphasic reaction system allowed high substrate loadings whereas the microreactor ensured excellent mass transfer rates between organic and aqueous phases. Finally, using a Teflon AF-2400 membrane in a tube-in-tube fashion allowed for radial delivery of oxygen and improved oxygen availability with respect to one point oxygen delivery in an aqueous/organic/air three phase segmented flow microreactor. Thereby a successful preparative scale biotransformation reaction in a segmented flow tube-in-tube reactor was possible yielding 740 mg of product purified by chromatography and subsequent sublimation. The STY of 14.5 g L total-1 h-1 reached is the highest reported for this enzyme. It is 38 times higher as compared to the batch reactions described earlier (Lutz et al. 2002). Oxygen transfer rates through Teflon AF-2400 membrane were as high as 24 mM min-1 i.e. 16 times higher than in traditional biotechnological processes in stirred-tank reactors. The presented reactor is a promising tool for oxygen dependent biocatalytic reactions in microreactors, and may be regarded as a basis for applications in gram scale organic biosyntheses. Furthermore, it may become a platform for other gas dependent reactions since it extends the operational boundaries beyond the scope of available reactors with respect to aqueous/organic mass transfer and gas transfer rates.|
|Subject Headings (RSWK):||Enzym|
|Appears in Collections:||Lehrstuhl Biotechnologie|
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