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

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

Supported liquid membrane as a novel tool for driving the equilibrium of ω-transaminase catalyzed asymmetric synthesis.

An attractive option to produce chiral amines of industrial importance is through asymmetric synthesis using ω-transaminase. However, reaching high yields often requires a strategy for shifting the equilibrium position. This paper describes a novel strategy for handling this problem. It involves the use of a supported liquid membrane (SLM) together with a packed bed reactor. The reactor contains Escherichia coli cells with ω-transaminase from Arthrobacter citreus, immobilized by flocculation with chitosan. The SLM consists of a hollow fibre membrane contactor in which the pores contain undecane. The system enables continuous extraction of the amine product and was used to successfully shift the equilibrium in asymmetric synthesis of (S)-α-methylbenzylamine (MBA). A conversion of 98% was reached, compared to 50% without product extraction. Moreover, a selective extraction of the produced MBA was realized. A high product concentration of 55g/l was reached after 80h, and the system showed promising potential for continuous operation

Chitosan flocculation: An effective method for immobilization of E. coli for biocatalytic processes.

Immobilization of Escherichia coli cells containing a ω-transaminase was carried out by flocculation with chitosan and the preparation was used in asymmetric synthesis of (S)-4'-cyano-α-methylbenzylamine, recycled in five consecutive batches. Chitosans with different molecular weights and degrees of acetylation were compared and effects of varying the chitosan properties, cell concentration and ratio of cells to chitosan were studied. Immobilization was achieved by increasing the pH to slightly alkaline, which induced the formation of large fast sedimenting flocs. Although an effective immobilization was obtained using most types of chitosan, high molecular weight and low degree of acetylation were considered favourable properties, resulting in good floc stability and quick sedimentation. It was found that it was possible to affect the floc characteristics, by changing the ratio of cells to chitosan in such a way that preparations resembling either entrapped or cross-linked cells could be obtained. The volume of the sedimented preparation decreased approximately 50% when increasing the cell to chitosan ratio from 2g/g to 10g/g at a constant amount of cells. Despite very high concentrations of cells (10-100g cells/g chitosan) in the flocculated preparations, diffusion limitations were minimal. Flocculation with chitosan was considered a simple and effective method for immobilization of E. coli cells for biocatalytic processes

An improved process for biocatalytic asymmetric amine synthesis by in situ product removal using a supported liquid membrane

Chiral amines are important building blocks in the pharmaceutical industry, and the biocatalytic synthesis of these compounds using ω-transaminases has been increasingly studied in recent years. In principal, asymmetric synthesis of chiral amines from a prochiral ketone is the preferable route, but it is often hampered by an unfavourable equilibrium position and product inhibition. An effective method for product removal is therefore necessary to drive the reaction towards product formation. In a recent study (Rehn et al., 2014) [29] we reported on the successful use of a supported liquid membrane (SLM) for the in situ product removal (ISPR) of (S)-α-methylbenzylamine (MBA) produced by Arthrobacter citreus ω-transaminase present in immobilized Escherichia coli cells. In the present work, we thoroughly discuss the factors influencing the performance of the SLM system and considerations for its successful use. Moreover, the system is further improved by implementing continuous control of the reactor pH using the amine donor substrate, and regeneration of the SLM unit at regular intervals to maintain the extraction performance, allowing the accumulation of 1.0 M (121 g/l) product in the stripping phase during operation for 91 h