The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.@en

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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Two non-canonical amino acids (ncAAs) with bio-orthogonal reactive groups, namely, p-azido-l-phenylalanine (p-AzF) and p-propargyloxy-l-phenylalanine (p-PaF), were genetically inserted into an aldo-keto reductase (AKR) and an alcohol dehydrogenase (ADH), respectively, at two preselected sites for each enzyme. The variants were expressed in the genome recoded bacterium Escherichia coli C321.ΔA. Supernatants of the individual cell lysates were subsequently mixed to produce orderly combi-crosslinked enzymes (O-CLEs) of AKR and ADH by co-polymerization of the two variants through their reactive bio-orthogonal groups. The site-specific cross-linked enzymes (S-CLEs) and cross-linked enzyme aggregates (CLEAs) were produced using dibenzocycloocta-4a,6a-diene-5,11-diyne (DBA) and glutaraldehyde as the crosslinking agent, respectively. The catalytic efficiencies of the O-CLEs, S-CLEs and combi-CLEAs were determined using the water soluble dihydro-4, 4-dimethyl-2, 3-furandione as a surrogate substrate in aqueous solution at 37 °C. The O-CLEs exhibited the highest catalytic efficiency (K_cat/K_M = 11.36 S⁻¹ mM⁻¹) that was 4.24 and 22.27 times that of S-CLEs and combi-CLEAs, respectively. In the asymmetric cascade synthesis of (R)-1-(2-chlorophenyl) ethanol the product yield after 14 h using the O-CLEs, S-CLEs and the combi-CLEAs was 93%, 55% and 16%, respectively. Moreover, high activities and selectivity (ee > 99.99%) were maintained at high substrate concentrations in prolonged operation.

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