Applying Enzymatic Cascades for ISCPR in ω-transaminase Systems

Biocatalysis complements the classical organic synthesis, and in many cases the superior selectivity of a biocatalyst is a strong driver explaining why there are an increasing number of processes where traditional organic synthesis has been replaced or combined with biocatalytic industrial process steps. An important fact is also that different types of selectivity make biocatalysis an excellent tool for overcoming difficulties typically associated with organic synthesis. Regioselectivity of the biocatalysts offers potential process simplification compared to the organic synthesis routes (reduction of the number of protective/deprotective steps), and stereoselectivity of the biocatalyst enables production of the desired chiral compounds, which often are building blocks of APIs. Currently there are many established processes in the industry using biocatalysis (≈ 300), e.g the usage of lipases, esterases, ketoreductases and proteases and many more emerging biocatalysts such are monoamine oxidases, transaminases and P450 monooxygenases to name a few. The focus of this thesis is the biocatalytic synthesis of small molecule pharmaceuticals (Mw<1000), and in particular the production of optically pure amines via ω-transaminases, which is an interesting class of reactions for the pharmaceutical industry. There are many challenges related to the realization and implementation of these technologies, and attempts of tackling them have been numerous. In some cases ω-transaminase catalyzed reactions are thermodynamically challenged and equilibrium shifting strategies are required. The proposed equilibrium shifting strategies are selection of an amino donor, excess of an amino donor, in-situ product removal (ISPR) and in-situ co-product removal. (ISCPR). For severely thermodynamically challenged reactions ISCPR by enzymatic cascades often provides the only viable option as equilibrium shifting strategy. In the literature several enzymatic cascades have been reported as an ISCPR for the ω-transaminase systems, however in most cases no process considerations have been made and the consequences of using a givens cascade in an industrial process context have thus not been considered properly. In this research lactate dehydrogenase (LDH) (E.C. 1.1.1.27), alanine dehydrogenase (E.C. 1.4.1.1) (AlaDH) and yeast alcohol dehydrogenase (E.C. 1.1.1.1) (YADH) have been researched as co-product degrading enzymes and glucose dehydrogenase (GDH) (E.C. 1.1.1.47) and formate dehydrogenase (E.C. 1.2.1.2) (FDH) as co-factor regeneration enzymes. Additionally pyruvate decarboxylase (E.C. 4.1.1.1) (PDC) and acetolactate synthase (E.C. 2.2.1.6) (ALS) have been considered as co-product degrading options. This work presents a procedure for cascade selection based on process considerations: thermodynamics, selectivity and operational stability while the final selection is further supported by the use of kinetic models. From the above presented cascade system options, the selection procedure identified the LDH/FDH cascade system as the system that is most promising for future industrial implementation. Furthermore, the required improvements of the ω-transaminase have been identified as a function of the added cascade enzymes and for the case γLDH = 11 g L-1, γFDH = 11 g L-1 and cNADH = 0.1 mmol L-1, it was found that the ω-transaminase activity expressed as Vmax_f,r is required to be 55.33 mmol min-1 L-1 to achieve 95 % conversion within 24 h. Further investigation concluded that a significant LDH concentration reduction is possible if inhibition by lactate is alleviated (preferably by protein engineering). This thesis identified the UFMR (UltraFiltration Membrane Reactor) as a viable process design option and charge analysis showed that ISPR is possible via ion exchange resins or electrodialysis. An ISPR example showed that process intensification could yield significant reductions in the required ω-transaminase activity improvement (up to five fold improvement) needed to achieve a viable industrial process, as well as reduction of required tolerance toward product inhibition. Although this thesis has been based on a specific case of a severely thermodynamically challenged ω-transaminase reaction (Keq = 4.03∙10-5), the selection framework can be transferred to any thermodynamically challenged reaction where the use of ISCPR by enzymes is considered to shift the equilibrium. Therefore, this work delivers: a) a method for initial investigation of thermodynamic limitations and viability of one or more equilibrium shifting strategies; b) a method for selecting a viable cascade option for ISCPR based on industrial conditions; c) information on the required enzyme performance e.g. the activity of the ω-transaminase, and potentially required compromises using process intensification tools and methods.