(438e) Multi-Enzymatic Cascade Reactions with Integrated Separation in a Complex 2-Phase System

In the field of white biotechnology the sustainable production of flavors and fragrances is increasingly gaining attention. Reactions with isolated enzymes allow for high selectivity and purity under mild reaction conditions, and less by-products, therefore enabling an overall sustainable production process. In this contribution a novel in-vitro multi-enzymatic cascade reaction system for the continuous and sustainable production of cinnamyl cinnamate in a 2-phase system is introduced.

Starting with cinnamyl aldehyde as substrate an alcohol dehydrogenase converts this to cinnamyl alcohol in a stirred tank reactor (reactor 1). A formate dehydrogenase ensures co-factor regeneration by the conversion of formate to CO₂. An extractive centrifugation allows for a reaction-integrated intermediate (cinnamyl alcohol) separation with an organic phase (separation), consequently enhancing the productivity of the dehydrogenases in the water-based buffer system in reactor 1. In the organic phase cinnamyl acid is added and a lipase is used to catalyze an esterification reaction resulting in cinnamyl cinnamate formation in a recycle packed-bed reactor (reactor 2).

The kinetic data of the two-substrate red-ox reactions catalyzed by the dehydrogenases were determined in the buffer system saturated with the organic solvent. A mathematical model was implemented. Experiments of the lipase-catalyzed esterification reaction of cinnamyl alcohol and cinnamyl acid to cinnamate were performed at the temperature optimum of the lipase (60°C). However, during extractive centrifugation, the organic phase is saturated with water, thereby decreasing the lipase activity.

Exemplary for the analyzed process parameters in pilot scale operation, the influence of the extraction temperature (separation) on the lipase activity (reactor 2) was evaluated. Lipase activity was increased by over 300% by decreasing the extraction temperature from 60°C to 15°C. Such increase can be explained by analyzing the water saturation concentration of the organic phase exiting the centrifuge. A temperature decrease of 75% during extraction results in a nearly linear corresponding lower water saturation concentration. The side product concentration prior to esterification is thereby decreased. These results show that further drying of the organic phase is desirable. Hereby, promising results were achieved using an additional integrated adsorption step. These results can be compared to an adsorption step prior to the lipase reaction.

In this contribution, the pilot plant set-up as well as experimental results are presented. Reaction kinetic data of all enzymes were determined. Experiments in pilot scale were performed, and process parameters were analyzed. Using ACM© from AspenTech, a computer model for the simulation of the cascade reaction is thereby introduced.