Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry

Sheldon, Roger A.; Wang, Anming; Xie, Tian; Li, Qiao; Zhang, Luo; Pei, Xiaolin; Wu, S. Z.; Chen, Haomin

doi:10.1039/d3gc01898a

“…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%

“…[17] In our previous work, we constructed a nano-reactor with a multienzyme assembly to synthesize chiral pharmaceutical intermediates by incorporating non-canonical amino acids (ncAAs) into enzymes. [18] In this work, we proposed a dualenzyme ordered coating strategy that combines the advantages of carrier and carrier-free immobilization. A dualenzyme cascade system was developed to synthesize (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone by regenerating the cofactor NADPH in situ.…”

Section: Introductionmentioning

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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“…Among these, crosslinking is the most promising technique that allows for the incorporation of enzymes and redox molecules onto the support platform [94]. In order to create the enzyme cascade system, the crosslinking technique is employed to co-immobilize different enzymes on the support surface with close proximity, thus allowing for its catalytic efficiency and reusability [95]. In addition, in some cases, the crosslinking technique can also control spatial distance and substrate channeling to attain catalytic efficiency [96].…”

Section: Crosslinked Supportmentioning

confidence: 99%

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells

Kalyana Sundaram,

Hossain,

Rezki

et al. 2023

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Nanomaterials, including carbon nanotubes, graphene oxide, metal–organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.

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

Luo,

Qiao,

Chen

et al. 2024

Angew Chem Int Ed

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2

0

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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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Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry

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...

Cited by 6 publications

References 85 publications

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

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

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells

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

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