Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin <i>via</i> strain-promoted alkyne–azide cycloaddition

Li, Huimin; Yin, Youcheng; Wang, Anming; Li, Ningning; Wang, Ru; Zhang, Jing; Chen, Xinxin; Pei, Xiaolin; Xie, Tian

doi:10.1039/c9ra09067c

Cited by 10 publications

(5 citation statements)

References 42 publications

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“…51 The specific protein expression and purification experimental method is derived from our previous work. 52 Plasmid preculture containing the akr multipoint mutant gene was obtained by overnight growth in LB, shaking at 34 °C with appropriate antibiotics (ampicillin, chloramphenicol, and kanamycin) added to LB to maintain the plasmid. The culture was diluted 100-fold and reexpanded in fresh LB containing antibiotics to expand the plasmid culture.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

Wang

et al. 2020

ACS Sustainable Chem. Eng.

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To develop a method for preparing rapidly, precisely, and bio-orthogonally cross-linked enzymes (RP-CLEs), nonstandard amino acids (NSAAs) were inserted into the enzyme protein, and microwave irradiation was used to accelerate its site-specific linkage through Cu-free strain-promoted alkyne–azide cycloaddition (SPAAC). To this end, we selected aldehyde ketone reductase (AKR) as model enzyme, and AKR mutants were obtained by five-point insertion of p-azido-l-phenylalanine (pAzF) which were subsequently cross-linked to form RP-CLEs from cell lysate supernatant under microwave irradiation. The AKR five-point mutant and corresponding RP-CLEs were characterized using MALDI-TOF MS, SEM, and FT-IR, respectively. The specific activities of RP-CLEs of three-point and five-point AKR were 1.27 and 2.06 U·mg–1, 1.21- and 2.16-fold those of the corresponding free enzymes, respectively. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol, the yield was up to 90.8%, and the ee was 99.98%. Moreover, after 6 consecutive 12 h reaction cycles, AKR five-point RP-CLEs still retained 80% of their initial activity. Thus, depending on the enzyme structure analysis, different numbers of NSAAs could be reasonably incorporated into the protein to accurately guide and control the covalent linkage to form RP-CLEs. This green method could be further developed both to generate bio-orthogonally cross-linked enzyme and separate them from nontarget proteins for industrial biocatalysis.

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Section: ■ Experimental Sectionmentioning

confidence: 99%

Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

Wang

et al. 2020

ACS Sustainable Chem. Eng.

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“…The effects of the incorporation number and site of the ncAAs on the loading and thermal stability of the immobilized AKR were determined. 526 They found that the half-life of the five-point immobilized AKR was 7-fold higher than that of the free enzyme at 60 °C. Immobilization may increase stability by mimicking the natural "microenvironmental habitat" of enzymes in living cells as most of the enzymes are attached to the cellular membranes, cellular organelles, and cytoskeleton.…”

Section: Enzyme Immobilization Using Ncaasmentioning

confidence: 99%

“…Recently, aldehyde ketone reductase (AKR) bearing multiple azide side chains was immobilized onto alkanylated epoxy resin using SPAAC. The effects of the incorporation number and site of the ncAAs on the loading and thermal stability of the immobilized AKR were determined . They found that the half-life of the five-point immobilized AKR was 7-fold higher than that of the free enzyme at 60 °C.…”

Section: Chemical Reactive Handles Of Ncaas For Enzyme Conjugationmentioning

confidence: 99%

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

et al. 2021

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The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

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“…Li et al also utilized 4-AzPhe, immobilizing aldehyde ketone reductase (AKR) variants to a bicyclononyne-functionalized resin. 368 Variants containing 4-AzPhe at one of five positions were tested, all of which displayed 5−16% increased activity upon immobilization and were also more thermostable than the free unmodified enzyme. Impressively, combining all five 4-AzPhe substitutions into one variant enhanced thermostability further, with over 70% residual activity recorded after 24 h incubation at 60 °C, compared to 5% for the free enzyme.…”

Section: Stabilization Via Noncovalent Interactionsmentioning

confidence: 99%

“…Additionally, the SPAAC-linked mutants were modestly more thermostable than the randomly cross-linked WT, with up to 1.2-fold higher residual activity recorded after a 29 h incubation at 50 °C. Li et al also utilized 4-AzPhe, immobilizing aldehyde ketone reductase (AKR) variants to a bicyclononyne-functionalized resin . Variants containing 4-AzPhe at one of five positions were tested, all of which displayed 5–16% increased activity upon immobilization and were also more thermostable than the free unmodified enzyme.…”

Section: Augmenting Functionmentioning

confidence: 99%

Noncanonical Amino Acids in Biocatalysis

Birch-Price,

Hardy,

Lister

et al. 2024

Chem. Rev.

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In recent years, powerful genetic code reprogramming methods have emerged that allow new functional components to be embedded into proteins as noncanonical amino acid (ncAA) side chains. In this review, we will illustrate how the availability of an expanded set of amino acid building blocks has opened a wealth of new opportunities in enzymology and biocatalysis research. Genetic code reprogramming has provided new insights into enzyme mechanisms by allowing introduction of new spectroscopic probes and the targeted replacement of individual atoms or functional groups. NcAAs have also been used to develop engineered biocatalysts with improved activity, selectivity, and stability, as well as enzymes with artificial regulatory elements that are responsive to external stimuli. Perhaps most ambitiously, the combination of genetic code reprogramming and laboratory evolution has given rise to new classes of enzymes that use ncAAs as key catalytic elements. With the framework for developing ncAA-containing biocatalysts now firmly established, we are optimistic that genetic code reprogramming will become a progressively more powerful tool in the armory of enzyme designers and engineers in the coming years.

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Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne–azide cycloaddition

Abstract: Stable immobilization of aldehyde ketone reductase mutants containing non-standard amino acids on an epoxy resin via strain-promoted alkyne–azide cycloaddition.

Cited by 10 publications

References 42 publications

Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

Noncanonical Amino Acids in Biocatalysis

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