Precision Engineering the Co‐Immobilization of Enzymes for Cascade Biocatalysis

The design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. Herein, the SpyTag/SpyCatcher binding pair was fused to the N‐terminus of an alcohol dehydrogenase (ADH) and an aldo‐keto reductase (AKR), respectively. A non‐canonical amino acid (ncAA), i.e. p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into pre‐selected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azido‐alkynyl for the immobilization of AKR and ADH enabled the sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to asymmetrically synthesize (S)‐1‐(2‐chlorophenyl) ethanol from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74% and good reproducibility, retaining 80% of its initial activity after six cycles. The product had an enantiomeric excess (e.e.) of 99.9% that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, approximately 1.7 times greater than that of the traditional single‐layer immobilization. More importantly, it simultaneously achieves both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.