Enzyme activity in nonaqueous media (e.g. conventional organic solvents) is typically lower than in water by several orders of magnitude. There is a rising interest of developing new nonaqueous ...solvent systems that are more “water-like” and more biocompatible. Therefore, we need to learn from the current state of nonaqueous biocatalysis to overcome its bottleneck and provide guidance for new solvent design. This review firstly focuses on the discussion of how organic solvent properties (such as polarity and hydrophobicity) influence the enzyme activity and stability, and how these properties impact the enzyme’s conformation and dynamics. While hydrophobic organic solvents usually lead to the maintenance of enzyme activity, solvents carrying functional groups like hydroxys and ethers (including crown ethers and cyclodextrins) can lead to enzyme activation. Ionic liquids (ILs) are designable solvents that can conveniently incorporate these functional groups. Therefore, we systematically survey these ether- and/or hydroxy-functionalized ILs, and find most of them are highly compatible with enzymes leading to high activity and stability. In particular, ILs carrying both ether and tert-alcohol groups are among the most enzyme-activating solvents. Future direction is to learn from enzyme behaviors in both water and nonaqueous media to design biocompatible “water-like” solvents.
Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a ...challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronological overview of the intertwined development of microfluidics droplet‐based compartmentalization methods and in vivo directed evolution of enzymes.
From bulk emulsification to on‐chip droplet formation and sorting, droplet microfluidics offers numerous tools for high‐throughput screening. Directed evolution has greatly benefitted from these technological advances over the last twenty years. This review article gives a chronological overview of in vivo directed evolution of enzymes facilitated by droplet microfluidics.
The enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal proline as main catalytic residue to facilitate several promiscuous C–C bond-forming reactions via enzyme-bound enamine ...intermediates. Here we show that the active site of this enzyme can give rise to further synthetically useful catalytic promiscuity. Specifically, the F50A mutant of 4-OT was found to efficiently promote asymmetric Michael additions of nitromethane to various α,β-unsaturated aldehydes to give γ-nitroaldehydes, important precursors to biologically active γ-aminobutyric acids. High conversions, high enantiocontrol, and good isolated product yields were achieved. The reactions likely proceed via iminium ion intermediates formed between the catalytic Pro-1 residue and the α,β-unsaturated aldehydes. In addition, a cascade of three 4-OT(F50A)-catalyzed reactions followed by an enzymatic oxidation step enables assembly of γ-nitrocarboxylic acids from three simple building blocks in one pot. Our results bridge organo- and biocatalysis, and they emphasize the potential of enzyme promiscuity for the preparation of important chiral synthons.
The endo-β-N-acetylglucosaminidases (ENGases) are an enzyme class (EC 3.2.1.96) produced by a range of organisms, ranging from bacteria, through fungi, to higher order species, including humans, ...comprising two-sub families of glycosidases which all cleave the chitobiose core of N-linked glycans. Synthetic applications of these enzymes, i.e. to catalyse the reverse of their natural hydrolytic mode of action, allow the attachment of N-glycans to a wide variety of substrates which contain an N-acetylglucosamine (GlcNAc) residue to act as an 'acceptor' handle. The use of N-glycan oxazolines, high energy intermediates on the hydrolytic pathway, as activated donors allows their high yielding attachment to almost any amino acid, peptide or protein that contains a GlcNAc residue as an acceptor. The synthetic effectiveness of these biocatalysts has been significantly increased by the production of mutant glycosynthases; enzymes which can still catalyse synthetic processes using oxazolines as donors, but which do not hydrolyse the reaction products. ENGase biocatalysts are now finding burgeoning application for the production of biologically active glycopeptides and glycoproteins, including therapeutic monoclonal antibodies (mAbs) for which the oligosaccharides have been remodelled to optimise effector functions.
•Redox enzymes enable the synthesis of chiral amines.•Monoamine oxidases (MAOs) catalyze aza-Friedel Crafts reactions and aromatisations.•Amine Dehydrogenases (AmDHs) catalyze the asymmetric ...amination of ketones.•Imine Reductases (IREDs) catalyse the asymmetric reduction of prochiral imines.•Reductive Aminases (RedAms) catalyse the formation of secondary amines.
Chiral amines feature in a large number of small molecule pharmaceuticals, and thus methods for their asymmetric synthesis are of considerable interest. Biocatalytic approaches have come to the fore in recent years as these offer advantages of superior atom economy, mild reaction conditions and excellent stereoselectivity. Advances in redox cofactor process technology have meant that oxidoreductase enzymes in particular now have growing potential as industrial catalysts for amine formation. In this review we survey recent developments in the discovery and application of oxidoreductase enzymes for amine production, including Monoamine Oxidases (MAOs), engineered and natural Amine Dehydrogenases (AmDHs), Imine Reductases (IREDs) and Reductive Aminases (RedAms), in addition to their application in enzyme cascades.
Impact experiments enable single particle analysis for many applications. However, the effect of the trajectory of a particle to an electrode on impact signals still requires further exploration. ...Here, we investigate the particle impact measurements versus motion using micromotors with controllable vertical motion. With biocatalytic cascade reactions, the micromotor system utilizes buoyancy as the driving force, thus enabling more regulated interactions with the electrode. With the aid of numerical simulations, the dynamic interactions between the electrode and micromotors are categorized into four representative patterns: approaching, departing, approaching‐and‐departing, and departing‐and‐reapproaching, which correspond well with the experimentally observed impact signals. This study offers a possibility of exploring the dynamic interactions between the electrode and particles, shedding light on the design of new electrochemical sensors.
Biocatalytic metal–organic framework micromotors with controllable vertical motion are applied to electrochemical impact measurements, which offer a possibility of exploring the dynamic interactions between the electrode and the particles and shed light on the design of new electrochemical sensors.
G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been ...widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3' end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3' adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid-base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions.
Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial ...applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.
Chiral sulfones are recurrent motifs in pharmaceuticals and bioactive molecules. Although chemical methods have been developed to afford α‐ or β‐ chiral sulfones, these protocols rely heavily on the ...pre‐synthesis of structurally complicated starting materials and chiral metal complexes. Herein, we described a photoenzymatic approach for the radical‐mediated stereoselective hydrosulfonylation. Engineered variants of ene reductases provide efficient biocatalysts for this transformation, enabling to achieve a series of β‐chiral sulfonyl compounds with high yields (up to 92 %) and excellent e.r. values (up to 99 : 1).
A photoenzymatic strategy for radical‐mediated stereoselective hydrosulfonylation has been developed. An engineered OYE1 enzyme enabled the generation of a series of β‐chiral sulfonyl compounds in high yields (up to 92 %) with excellent e.r. values (up to 99 : 1).
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