*3.1. Salt Solubilities*

The solubility difference between both salts, donor salt and product salt, is the main parameter within the shown reaction mode. The donor salt must have a significantly higher solubility than the product salt, which will only then crystallize selectively from solution. Using the 3,3-diphenylpropionate (3DPPA) as the anion results in a solubility difference of approx. 50 mM between the donor salt (IPA-3DPPA) and the product salt (3MPEA-3DPPA) (Figure 3). For the investigated biocatalytic reaction system the concentration of the donor salt remains for above pH 7 and 30 ◦C, at >50 mM, while the product salt is considerably less soluble at approx. 5 mM, depending on the chosen pH in solution (Figure 3A). These results are comparable with the model product amine salt 1-phenylethylammonium 3,3-diphenylpropionate in an earlier study [30]. Please note that the shown concentrations may be altered by the presence of other salts such as other buffer components, impurities and especially the additionally used isopropylamine. Changes in temperature will also affect the solubilities of these two main salts, however the observed effect is relatively small. As shown in Figure 3B at pH 7.5 no significant effect is visible and the donor salt remains strongly more soluble than the product amine salt. The choice of temperature is fortunately mostly controlled by the temperature optimum of the biocatalyst itself, which limits the choice of reaction temperature to a narrow range at 30 ◦C.

**Figure 3.** pH- and temperature-dependent solubility profile of the donor salt IPA-3DPPA and product salt 3MPEA-3DPPA; (**A**) pH dependency in 50 mM phosphate buffer at 30 ◦C; (**B**) temperature dependency in 25 mM HEPES buffer pH 7.5.

### *3.2. Single Membrane Reactor*

The central membrane reactor is the key component within the triple vessel concept. The applied membrane primarily retains the biocatalyst (whole *E. coli* cells) in the biocatalyst chamber from the remaining solution and both solid salts, IPA-3DPPA and 3MPEA-3DPPA, in the salt chamber (Figure 4). During the reaction, IPA-3DPPA is continuously consumed as the amount of product salt increases and eventually accumulates as the only solid phase.

The dissolved reactants diffuse freely between both chambers and a relevant diffusion limitation was not observed (full equilibrium conditions can be achieved within ca. 10 min). This approach provides an alternative to classical encapsulation and immobilization approaches and thus prevents undesired deactivation or diffusion problems of the biocatalytic reaction system [32–36]. The applied PVDF transfer membrane is fully biocompatible and was described by Wachtmeister et al. in 2014 for a lyase-catalyzed reaction [37]. In addition, the use of a membrane reactor offers a simple adjustment of the biocatalytic synthesis system without interfering with the solid salt phases, including the addition or a full exchange of the biocatalyst during the reaction. In addition, the accumulation of inactivated biocatalyst in combination with an undesired mixing with the product salt is prevented, which simplifies downstream processing enormously to a simple filtration step.

**Figure 4.** Reaction flow in the membrane reactor.

As shown in Figure 5, a batch experiment of the membrane reactor, without any connection to a saturator and crystallizer, allows the conversion of 55 mM 3MAP to the corresponding product amine salt 3MPEA-3DPPA. The product salt accumulates exclusively in the salt chamber, while the donor salt IPA-3DPPA is consumed in parallel in the salt chamber. A small amount of the product amine is always present in the biocatalyst chamber, which relates to the solubility limit of the product salt in solution. Eventually the reaction stops at approx. 55 mM product concentration due to an accumulation of acetone in the aqueous phase due to the absence of an active acetone removal step, which equals the equilibrium position of this biochemical reaction. Due to absence of an any observable substrate and product inhibition at the chosen reaction conditions, the only rate determining step is the available catalytic activity of the biocatalyst, which leaves a lot of room for optimization. Transport through the membrane and crystal growth are always significantly faster and did not limit the overall process.

**Figure 5.** 3MPEA concentration curve in the chambers of the membrane reactor, 30 ◦C, 25 mM HEPES buffer pH 7.5, 100 mM 3MAP, 100 mM IPA-3DPPA, 100 mM additional isopropylamine and 5 mM PLP (within entire solution).
