Process development, analysis and improvement for biphasic oxidoreductions on miniplant scale

The biocatalytic conversion of hydrophobic substrates indicates the application of nonpolar solvents in an aqueous/organic two phase system. The substrate can be dissolved in high concentrations within the organic bulk phase and diffuses, according to the partition equilibrium into the aqueous phase, where it is converted. A hydrogel-stabilized aqueous phase is capable of protecting the enzymes from surface deactivation and hydrodynamic stress. The reduction of acetophenone catalyzed by the carbonyl reductase from Candida parapsilosis (CPCR2) and the alcohol dehydrogenase from Lactobacillus brevis (LbADH G37D) is a model reaction for the conversion of hydrophobic substrates. The co-factor NADH is regenerated in situ by oxidizing formic acid, catalyzed by formate dehydrogenase from Candida boidinii (FDH). Aim of this work was to develop, establish and analyze options for process control, enabling stable oxidoreductions without pH-increase by oxidized formic acid. Further, the prerequisites for establishing the two processes, a reliable enzyme supply and an appropriate immobilization technique were addressed. In order to discover alternative methods to cryo-gelation of polyvinylic alcohol (PVA) for the hydrogel entrapment of enzymes, due to low residual activities obtained for CPCR2 and FDH and limited protein capacity, CPCR2 and FDH were immobilized by different methods. The immobilization within glutaraldehyde-cross-linked polyallylamine (PAAm) was applied successfully. Methylene-bis-acrylamide-(BIS)-cross-linked poly-N-isopropyl amide (PNIPAAm) only lead to a residual activity of FDH. An existing method for cryo-gelation of PVA for immobilization of enzymes within defined (PVA) hydrogel beads was adapted, established and characterized for the application within this work. A controlled process in a two phase reaction system was developed for the reduction of the hydrophobic model substrate acetophenone dissolved in n-heptane to 1-phenylethanol catalyzed by CPCR2, immobilized in a PVA hydrogel. After an experimental proof of the process concept, simulation studies with a holistic process model were performed in order to identify limitations for a rational improvement and to point out the potential of the process. As the measurement of CO2 produced during co-factor regeneration occurs time shifted, depending on the CO2 solubility within the immobilisates and the organic bulk phase, volume of the CO2-sink, temperature and CO2 concentrations within the dissolving phases, a more direct measurement technique, especially for high reaction rates, is indicated. To this end, dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online-monitoring of the pH-value within the immobilisates during the reaction, allowing a controlled dosage of formic acid. An experimental proof of concept could maintain a reaction, resulting in an increased substrate conversion, compared to the CO2-based control. In addition to the identification of promising immobilization techniques for application in aqueous/organic two phase systems, although with potential for optimization, it was shown that the two described process concepts constitute promising alternatives for controlled, biocatalytic, biphasic processes and model based process analysis can help revealing critical aspects and optimization potential