Continuous flow biocatalysis for citronellal synthesis using immobilized ene-reductases

In recent years, biocatalysis has emerged as a more sustainable alternative to traditional chemical catalysis, as enzymes work under mild reaction conditions, are highly selective and can be produced from renewable resources. However, when we consider their use on an industrial scale several drawbacks arise, mainly for the fact that enzymes are built to work in a biological environment with low substrate concentration, and tight regulation mediated by substrate or product. In addition, when isolated from cells, enzymes are usually unstable and their recycle is difficult. A possible way to overcome these limitations is to immobilize them and turn them into heterogeneous catalysts, allowing their reuse for either subsequent reactions, or continuous flow applications with immediate separation of the product from the reaction mixture [1]. We focused on the covalent co-immobilization of a suitable ene-reductase (ER, E.C. 1.6.99.1) and formate dehydrogenase (FDH, E.C. 1.17.1.9) as the cofactor-regenerating enzyme, to perform the enantioselective reduction of natural citral (6:4 mixture of geranial and neral) to either (R)- or (S)- enriched citronellal in a continuous flow reactor. The (R)-enantiomer is relevant as a precursor to (-)- menthol, while (S)-citronellal is used for fragrances synthesis [2]. The immobilization was performed with OYE2 from Saccharomyces cerevisiae to produce enriched (R)-citronellal, with NemA from Escherichia coli for enriched (S)-citronellal, and with an engineered NADP+-dependent FDH from Pseudomonas sp. 101 as the cofactor regenerating enzyme. The scaffold of choice was Seplife® EMC7014 epoxy resin to ensure high-density covalent bonds between the enzymes and the resin and lower leakage compared to immobilization based on weak interaction [3]. Initial studies in batch proved that the immobilized catalysts have a long shelf-life at 4 °C of over 2 months, and a high reusability as the conversion of 5 mM citral remains unchanged (80 % by GC-MS in 4 h) after six cycles. These data are promising for the use in either a packed-bed or a continuously stirred tank reactor with continuous substrate and cofactor feed, followed by separation of the product from the oxidized cofactor with membrane extraction to isolate citronellal and recirculate the cofactor