NADH regenerated using immobilized FDH in a continuously supplied reactor – Application to l-lactate synthesis

• Kinetic of the NADH regeneration using immobilized FDH was examined.
• Kinetic parameters of the enzymatic regeneration of NADH were determined.
• Optimization of the FDH immobilization by encapsulation, was achieved.
• We achieve 3 weeks of continuous synthesis of l-lactate using in situ regenerated NADH.
• We simulate the filter press reactor and propose NAD+ diffusivity into a chitosan polymer.

The present study deals with the design and optimization of a reduced-scale filter press reactor, containing an immobilized layer of formate dehydrogenase between two layers of chitosan, dedicated to the enzymatic synthesis of chiral molecules. Elaboration and optimization of the overall system was carried out to demonstrate the feasibility of the immobilization of formate dehydrogenase in a continuous synthesis process. The results demonstrate that the immobilized FDH keeps half of its enzymatic activity for practically two weeks. In addition, the polymeric matrix allows transfer of NAD+ with relatively high diffusion coefficients (2.1 × 10−11 m2 s−1). Experimental validation of NADH regeneration was achieved for pyruvate reduction to l-lactate. Simulation demonstrated that it is possible to achieve practically quantitative conversion of the NAD+ if the reaction channel reaches a length of half a meter.