**3. Results and Discussion**

Initial studies of the integrated use of crystallization in amine transaminase-catalyzed reactions included a direct application of a carboxylic acid, which yields the crystallization of the product amine salt but specifically avoids the crystallization of the donor amine isopropylamine as its salt (donor salt) [16]. This application of this concept results in a moderate apparent shift of the reaction equilibrium towards the product side but is unfortunately often limited to batch reactions and relatively low substrate concentrations due to a continuously increasing amount of solid product salt stopping the entire reaction.

In this study we present an alternative continuous approach towards this reactive crystallization, which intentionally includes the presence of the originally undesired donor amine salt isopropylammonium 3,3-diphenylpropionate. The donor amine salt dissolves continuously and thus release stoichiometric amounts of isopropylammonium and 3,3-diphenylpropionate into solution. Any excess beyond the solubility limit remains as a dispersed solid phase in the reaction mixture. This basically limits the amount of amine in solution to an absolute minimum, in contrast to conventional approaches using high excesses of isopropylamine in solution, which may cause limited enzyme stability [30,31]. Similarly, the substrate 3MAP is continuously dissolved in the solution up to its solubility limit, ensuring a constant 3MAP concentration in the aqueous solution throughout the process. Consequently, the reaction equilibrium in solution is based on the aqueous phase concentration since it is only accessible by the biocatalyst. The conversion towards the products leads to a continuous removal of the educts from the aqueous solution, which is adjusted to the original concentration due to the above mentioned solubility equilibrium. The reaction cycle is closed by the final continuous crystallization of the product amine salt, which removes in stoichiometric amounts the dissolved 3,3-diphenylpropionate anion. The only byproduct is acetone, which evaporates quite easily from solution due to its high vapor pressure at 30 ◦C [30]. Applications at large scale will require additional solutions to remove acetone effectively from solution to avoid a full stop of the in situ-product crystallization and the inhibition of the biocatalyst, for example, via stripping with an inert gas.

The presence of two solid salt phases requires a separation into two vessels to avoid an undesired mixing. In this work we present a triple vessel system, which separates both solid phases and the catalyst from each other, which enables the above mentioned continuous reaction mode (Figure 2). A membrane reactor is applied to retain the biocatalyst (amine transaminase from *Ruegeria pomeroyi*) behind a polyvinylidene fluoride membrane (PVDF), while the filtered mother liquor is pumped via peristaltic pumps through a crystallizer for product salt crystallization, a saturator for donor salt saturation and eventually back into the membrane reactor to close the loop. An exception is the connection between the membrane reactor and the crystallizer, which is directly fed by an overflow from the higher positioned membrane reactor.
