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).