Print Email Facebook Twitter Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry Title Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry Author Qiao, Li (Hangzhou Normal University) Luo, Zhiyuan (Hangzhou Normal University; Shanghai University of Traditional Chinese Medicine) Wang, Ru (Hangzhou Normal University) Pei, Xiaolin (Hangzhou Normal University) Wu, Shujiao (Hangzhou Normal University) Chen, Haomin (Hangzhou Normal University) Xie, Tian (Hangzhou Normal University; Shanghai University of Traditional Chinese Medicine) Sheldon, R.A. (TU Delft BT/Biocatalysis; University of Witwatersrand) Wang, Anming (Hangzhou Normal University) Date 2023 Abstract 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 (Kcat/KM = 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. To reference this document use: http://resolver.tudelft.nl/uuid:efe55fd8-7f5e-454d-840b-f84e8504c696 DOI https://doi.org/10.1039/d3gc01898a ISSN 1463-9262 Source Green Chemistry, 25 (19), 7547-7555 Part of collection Institutional Repository Document type journal article Rights © 2023 Li Qiao, Zhiyuan Luo, Ru Wang, Xiaolin Pei, Shujiao Wu, Haomin Chen, Tian Xie, R.A. Sheldon, Anming Wang Files PDF d3gc01898a.pdf 1.62 MB Close viewer /islandora/object/uuid:efe55fd8-7f5e-454d-840b-f84e8504c696/datastream/OBJ/view