Figure 1. Multi-enzyme cascade reaction sequence for the production of cinnamyl cinnamate from cinnamyl aldehyde with integrated co factor regeneration and in situ intermediate extraction in a two-phase system.
An alcohol dehydrogenase (ADH) converts cinnamyl aldehyde to cinnamyl alcohol in a buffer phase (0.1 M potassium phosphate buffer at pH 8.0) using the cofactor NADH. Integrated cofactor regeneration within this cascade reaction sequence is ensured through addition of a formate dehydrogenase (FDH) converting formate to CO2 under NAD+ consumption. After in situ extraction of the intermediate cinnamyl alcohol with xylene as an organic solvent and cinnamic acid addition, the lipase Novozym® 435 performs an esterification reaction in the organic phase to synthesize cinnamyl cinnamate and water. This process has successfully been established in both the laboratory and the miniplant.
Figure 2 shows the flow chart of the miniplant used for this three-enzyme cascade reaction. The two cofactor-coupled dehydrogenases are immobilized on silica particles according to Engelmann et al.and placed in a two-liter continuously operated stirred tank reactor tempered to 30 °C, equipped with a SpinChem® RBR S2 rotating bed reactor (SpinChem AB, Umeå, Sweden), spinning at 400 rpm (figure 2, A). A buffer tank is used for the organic phase and for cinnamic acid feeding (figure 2, B). According to the principles of process integration, the extraction step connects both phases as they are mixed and immediately separated in one apparatus, a CINC CS 50 extractive centrifuge (CINC Deutschland GmbH & Co. KG, Brakel, Germany) (figure 2, C). For the final esterification reaction step, the cinnamic acid- and alcohol-loaded xylene phase is subsequently pumped through a fixed bed reactor, containing 12.8 g of Novozym®435 and tempered to 60 °C according to the enzyme provider’s information (figure 2, D).