Results and Discussion

Optimization of the Enzymatic Imine Reductase Reaction

Initial experiments targeted an increase of the substrate concentration to facilitate higher productivities within the biocatalytic process using the highly enantioselective imine reductase fromPaenibacillus elgii B69. Considering the chosen conditions, the starting substrate concentration leads to a full conversion in the range of 25 mmol·L‑1 to 150 mmol·L-1, but a deactivation of the catalyst was found at even higher substrate concentrations (see Figure 2A). This is solely caused by a substrate inhibition and not a pH-shift due to the cofactor regeneration-based formation of gluconic acid by glucose dehydrogenase. Noteworthy, attempts using an excessive pH control with a 1 mol·L-1 phosphate buffer pH 7.5 were also not successful due to a complete deactivation of the biocatalyst. A full conversion was reached within 24 h under the chosen reaction conditions (Figure 2B). The final optimized reaction conditions facilitated full conversions at 30 °C, a substrate concentration of 150 mmol·L-1 and 500 mmol·L-1 of d‑glucose. These experiments clearly indicate that further improvements of the catalytic robustness towards higher substrate loadings and higher specific activities are required to improve the overall process productivity, which was not the main aim of this study. Other noticeable examples of imine reductase-catalyzed reactions at larger scale were recently reported for the biocatalytic reductive amination reaction between cyclohexanone and cyclopropylamine with an excellent volumetric productivity of 12.9 g·L-1·h-1 and a TON above 48000