ω-Transaminase Catalyzed Synthesis of Chiral Amines - Process Improvements Through Whole-cell Immobilization and in situ Product Removal

Abstract

The industrial use of biocatalysts is increasingly important in the production of e.g. pharmaceuticals, chemicals, fuels and foodstuffs. A successful biocatalytic process is dependent on many factors, such as the biocatalyst properties, the nature of the reaction, reactor type and downstream processing. This work is focused on two important issues: Immobilization of the biocatalyst and in situ product removal. Different methods were evaluated for immobilization of the biocatalyst, E. coli cells containing a recombinantly expressed ω-TA from A. citreus. It was found that flocculation with chitosan, enhanced by a pH adjustment, was a very simple and effective method, allowing high biocatalyst loading while maintaining good diffusion properties. The flocculated cells proved useful in both stirred tank and packed bed reactors. The process considered is the production of chiral amines, which are highly important building blocks in e.g. pharmaceuticals and agrochemicals. The use of ω-transaminases (ω-TAs) for the production of chiral amines has been a rapidly growing field of research during recent years. Asymmetric synthesis is the principally advantageous route because of the high theoretical yield without need for additional reaction steps. However, the equilibrium position is often unfavourable for the amine synthesis. Shifting the equilibrium is therefore a frequently addressed issue. A commonly studied model reaction, conversion of acetophenone to optically pure methylbenzylamine, was employed in this work, using isopropylamine (IPA) as the amine donor. In this work, in situ product removal was realized by using a supported liquid membrane (SLM). The SLM consisted of n-undecane present in the pores of a hollow fibre allowing three-phase extraction (aqueous : organic : aqueous). The bioreactor effluent was circulated on the one side of the membrane and an acidic stripping phase, capturing the amine product, on the other side. Selective extraction of the amine product, (S)-α-methylbenzylamine, was achieved due to its considerably higher hydrophobicity and its lower pKa value compared to IPA. Thus, similar concentrations of the two amines were extracted despite using a large excess of IPA to drive the reaction. A highly concentrated product, 98 g/l (810 mM), was obtained in 36 hours when using the SLM extraction system coupled to a stirred tank reactor. Ketones are not trapped in the stripping phase. Finally, parameters influencing the product extraction were investigated and discussed. The use of an SLM system was considered a valuable addition to other existing methods for improving ω-TA catalyzed asymmetric synthesis