Controlled chemical assembly of enzymes in cell lysate enabled by genetic-encoded nonstandard amino acids

Zhang, Jing; Wang, Ru; Luo, Zhiyuan; Jia, Dongmei; Chen, Haoming; Xiao, Qinjie; Zhang, Pengfei; Pei, Xiaolin; Wang, An Ming

doi:10.1039/d1qm01285a

Cited by 6 publications

(4 citation statements)

References 79 publications

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“…Based on our previous studies, the mutant sites for AKR were identified as 130S, 162Q, 189Q, and 232W, and these sites were 104A, 125I, 155Y, 199P, and 204A for ADH. [18,23] The selection principles for mutation sites are as follows. 1) far from the active center; 2) less impact on the protein structure, considering similar group size or electronegativity; 3) located on the surface of a protein.…”

Section: Construction Of Fused Enzymes and Their Mutantsmentioning

confidence: 99%

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

Luo,

Qiao,

Chen

et al. 2024

Angewandte Chemie

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The design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. Herein, 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), i.e. p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into pre‐selected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azido‐alkynyl for the immobilization of AKR and ADH enabled the sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to asymmetrically synthesize (S)‐1‐(2‐chlorophenyl) ethanol 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 an enantiomeric excess (e.e.) of 99.9% that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, approximately 1.7 times greater than that of the traditional single‐layer immobilization. More importantly, it simultaneously achieves both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

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Section: Construction Of Fused Enzymes and Their Mutantsmentioning

confidence: 99%

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

Luo,

Qiao,

Chen

et al. 2024

Angewandte Chemie

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“…In the biocatalytic asymmetric reduction of aryl alkyl ketones 93 the reductases have to cope with a wide variety of substituents in the aromatic ring, both electron-donating ( e.g. alkyl, alkoxy, amine), and electron-withdrawing ( e.g.…”

Section: Scope In the Enantioselective Synthesis Of Chiral Alcoholsmentioning

confidence: 99%

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

et al. 2023

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“…Here, if the insertion sites of NSAAs are rationally designed in space (Fig. 19(C)), the protein rings can be obtained 209 and further used as protein-based biomaterials for drug delivery or directly as clinical protein.…”

Section: Bio-orthogonal Immobilisation Mediated By Non-standard Amino...mentioning

confidence: 99%

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry

Pei

Luo

et al. 2022

Chem. Soc. Rev.

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Controlled chemical assembly of enzymes in cell lysate enabled by genetic-encoded nonstandard amino acids

Abstract: Enzyme proteins are nanometer-sized molecules with a three-dimensional structure that can be manipulated and assembled into highly ordered nanostructures, which allows access to advanced biological materials. Here, genetically-encoded nonstandard amino...

Cited by 6 publications

References 79 publications

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

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

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry

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