An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies

Optimal performance of multi‐step enzymatic one‐pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two‐step one‐pot recycling cascade utilizing the thiamine diphosphate (ThDP)‐dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) and the alcohol dehydrogenase from Ralstonia sp. (RADH) for the production of the vicinal 1,2‐diol (1R,2R)‐1‐phenylpropane‐1,2‐diol (PPD) using both enzymes as catalytically active inclusion bodies (CatIBs). PfBAL is hereby used to convert benzaldehyde and acetalydehyde to (R)‐2‐hydroxy‐1‐phenylpropanone (HPP), which is subsequently converted to PPD. For recycling of the nicotinamide cofactor of the RADH, benzyl alcohol is employed as co‐substrate, which is oxidized by RADH to benzaldehyde, establishing a recycling cascade. In particular the application of the RADH, required for both the reduction of HPP and the oxidation of benzyl alcohol in the recycling cascade is challenging, since the enzyme shows deviating pH optima for reduction (pH 6–10) and oxidation (pH 10.5), while both enzymes show only low stability at pH>8. This inherent stability problem hampers the application of soluble enzymes and was here successfully addressed by employing CatIBs of PfBAL and RADH, either as single, independently mixed CatIBs, or as co‐immobilizates (Co‐CatIBs). Single CatIBs, as well as the Co‐CatIBs showed improved stability compared to the soluble, purified enzymes. After optimization of the reaction pH, the RADH/PfBAL ratio and the co‐solvent content, we could demonstrate that almost full conversion (>90%) was possible with CatIBs, while under the same conditions the soluble enzymes yielded at most >50% conversion. Our study thus provides convincing evidence that (Co‐)CatIB‐immobilizates can be used efficiently for the realization of cascade reactions, i. e. under conditions where enzyme stability is a limiting issue