Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

The design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. In this study, 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), p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide–alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to synthesize (S)‐1‐(2‐chlorophenyl)ethanol asymmetrically 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 99.9 % ee, which that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single‐layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis. A double‐layered coating approach was developed for the precise and sequential immobilization of dual‐enzyme systems on resin particles using two biorthogonal counterparts of SpyTag/SpyCatcher and azide–alkyne cycloaddition. The method enables the combined purification and the ordered immobilization of enzymes and significantly enhances the catalytic efficiency and recyclability of multienzyme systems.