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.

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.

The covalent immobilisation of enzymes generally involves the use of highly reactive crosslinkers, such as glutaraldehyde, to couple enzyme molecules to each other or to carriers through, for example, the free amino groups of lysine residues, on the enzyme surface. Unfortunately, such methods suffer from a lack of precision. Random formation of covalent linkages with reactive functional groups in the enzyme leads to disruption of the three dimensional structure and accompanying activity losses. This review focuses on recent advances in the use of bio-orthogonal chemistry in conjunction with rec-DNA to affect highly precise immobilisation of enzymes. In this way, cost-effective combination of production, purification and immobilisation of an enzyme is achieved, in a single unit operation with a high degree of precision. Various bio-orthogonal techniques for putting this precision and elegance into enzyme immobilisation are elaborated. These include, for example, fusing (grafting) peptide or protein tags to the target enzyme that enable its immobilisation in cell lysate or incorporating non-standard amino acids that enable the application of bio-orthogonal chemistry.

The use of engineered ketoreductases (KREDS), both as whole microbial cells and isolated enzymes, in the highly enantiospecific reduction of prochiral ketones is reviewed. The homochiral alcohol products are key intermediates in, for example, pharmaceuticals synthesis. The application of sophisticated protein engineering and enzyme immobilisation techniques to increase industrial viability are discussed.