article · Angewandte Chemie International Edition
Combining multiple enzymes systematically onto solid supports is difficult. Researchers have engineered an orderly, layered co-immobilisation system for cascade biocatalysis using two enzymes, an aldo-keto reductase and an alcohol dehydrogenase. By integrating SpyTag/SpyCatcher binding pairs with the site-specific genetic insertion of an unnatural amino acid for azide-alkyne click chemistry, the enzymes were sequentially attached to porous microspheres. This structured dual-enzyme reactor was evaluated in the asymmetric synthesis of (S)-1-(2-chlorophenyl)ethanol from a prochiral ketone, allowing the simultaneous in situ regeneration of the cofactor NADPH. The reactor achieved a 74 percent conversion rate and delivered product with 99.9 percent enantiomeric excess, retaining 80 percent of its initial activity across six operational cycles. The layered method increased enzyme loading by roughly 1.7 times compared to conventional single-layer immobilisation while allowing concurrent purification and immobilisation.
Multi-enzyme cascades can produce valuable chiral chemicals cleanly, but attaching multiple fragile biocatalysts to solid supports often compromises efficiency and requires separate, costly purification steps. This layered immobilisation technique simplifies production by purifying and capturing enzymes in one operation. It produces durable, reusable catalytic beads that maintain high selectivity and activity, offering a more efficient platform for complex chemical syntheses.
This method is relevant to fine chemical and pharmaceutical manufacturers producing chiral alcohol intermediates that require cofactor recycling. The approach could benefit industrial biocatalysis teams seeking to streamline enzyme purification and lower production costs through catalyst reusability. Based on the abstract, the technology is applied and tested at laboratory scale on a single model reaction, meaning scaling, carrier durability, and performance on diverse substrates remain to be demonstrated.
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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.
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DOI: 10.1002/anie.202403539
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