Biocatalysis Research Articles

Intensified, Kilogram-Scaled, and Environment-Friendly: Chemoenzymatic Synthesis of Bio-Based Acylated Hydroxystyrenes.

Lignin-derived styrene derivatives are versatile building blocks for the manufacture of biobased polymers. As shown previously, phenol-protected hydroxystyrenes are accessible under industrially sound conditions (>100 g L-1, >95% yield) by subjecting biogenic phenolic acids to enzymatic decarboxylation and base-catalyzed acylation in nonaqueous media (wet cyclopentyl methyl ether, CPME). Herein, we demonstrate the production of 1 kg of 4-acetoxy-3-methoxy-styrene in a 10 L reactor and present practical adjustments to the up- and downstream processing that warrant a straightforward process and high isolated yields. Additionally, an environmental assessment is conducted, starting with a thorough E factor analysis to identify the sources that contribute most to the environmental burden (solvent and downstream processing). Also, the total CO2 production of the process is studied, including contributions from energy use and the treatment of generated wastes. The energy impact is evaluated through thermodynamic analysis, and the environmental footprint contributions by wastes-organic and aqueous fractions-are assessed based on CO2 emissions from solvent incineration and wastewater treatment, respectively. Overall, the holistic assessment of the process, its optimization, scale-up, product isolation, and environmental analysis indicate the feasibility of multistep chemoenzymatic reactions to deliver high-volume, low-value chemicals from biorefineries.

Deracemization of Sulfoxides Combining Electrocatalysis and Biocatalysis

Comprehensive SummaryThe concurrent implementation of cascade reactions that combine biocatalysis and chemocatalysis is a challenging undertaking. Electrocatalysis provides versatile catalytic abilities and allows for mild reaction conditions, offering the potential for designing concurrent chemoenzymatic cascade reactions. The research on bioelectrocatalysis has primarily concentrated on utilizing electrocatalysis to achieve cofactor regeneration of enzymes. In contrast with previous reports, herein, we developed a deracemization strategy involving the concurrent combination of biocatalytic reduction and anodic oxidation in an undivided cell to achieve chiral sulfoxides, demonstrating the good compatibility. We anticipate this study will offer an alternative pathway for the design of the cascade reaction combined with electrocatalysis and biocatalysis.

One-pot chemo- and photo-enzymatic linear cascade processes.

The combination of chemo- and photocatalyses with biocatalysis, which couples the flexible reactivity of the photo- and chemocatalysts with the highly selective and environmentally friendly nature of enzymes in one-pot linear cascades, represents a powerful tool in organic synthesis. However, the combination of photo-, chemo- and biocatalysts in one-pot is challenging because the optimal operating conditions of the involved catalyst types may be rather different, and the different stabilities of catalysts and their mutual deactivation are additional problems often encountered in one-pot cascade processes. This review explores a large number of transformations and approaches adopted for combining enzymes and chemo- and photocatalytic processes in a successful way to achieve valuable chemicals and valorisation of biomass. Moreover, the strategies for solving incompatibility issues in chemo-enzymatic reactions are analysed, introducing recent examples of the application of non-conventional solvents, enzyme-metal hybrid catalysts, and spatial compartmentalization strategies to implement chemo-enzymatic cascade processes.

Tailor-Made Bioactive Papers by Site-Specific and Orthogonal Covalent Immobilization of Proteins.

A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality. Enhanced green fluorescent protein (EGFP) was used as a model protein to allow quantification of protein loading via fluorescence imaging. Two approaches were identified that showed significantly increased loading efficiencies compared with the previously applied conjugation strategy. Additionally, all four methods were proven orthogonal to each other, allowing simultaneous immobilization of a mixture of proteins to a premodified assembly of two paper sheets.

A Strep-Tag Imprinted Polymer Platform for Heterogenous Bio(electro)catalysis.

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surface-sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.

A Self‐regulated Process from Bench to Pilot Production of Non‐natural Heterocycles Using Halohydrin Dehalogenases.

Halohydrin dehalogenases (HHDHs) have demonstrated significant potential as biocatalysts for synthesizing industrial heterocyclic compounds and chiral alkanolamines from aromatic substrates with NaOCN that are not feasible through chemosynthesis. To further study the HHDH reaction in real industrial applications, a diverse panel of HHDHs has been screened for their ability to convert aliphatic cyclohexene oxide. By using the best HHDH variant in form of a whole cell biocatalyst, a novel chemical process, regulated by the cascaded chemoenzymatic reactions for pilot‐scale ring‐opening reactions, has been successfully developed. Using the best HHDH hit in whole‐cell form provides a viable bio‐catalytic approach for the synthesis of related fine chemicals. Our research provides a promising strategy for scaling up non‐native ring‐opening reactions from the laboratory to industrial applications.

Nanoarchitectonics with a Membrane-Embedded Electron Shuttle Mimics the Bioenergy Anabolism of Mitochondria.

Enhanced bioenergy anabolism through transmembrane redox reactions in artificial systems remains a great challenge. Here, we explore synthetic electron shuttle to activate transmembrane chemo-enzymatic cascade reactions in a mitochondria-like nanoarchitecture for augmenting bioenergy anabolism. In this nanoarchitecture, a dendritic mesoporous silica microparticle as inner compartment possesses higher load capacity of NADH as proton source and allows faster mass transfer. In addition, the outer compartment ATP synthase-reconstituted proteoliposomes. Like natural enzymes in the mitochondrion respiratory chain, a small synthetic electron shuttle embedded in the lipid bilayer facilely mediates transmembrane redox reactions to convert NADH into NAD+ and a proton. These facilitate an enhanced outward proton gradient to drive ATP synthase to rotate for catalytic ATP synthesis with improved performance in a sustainable manner. This work opens a new avenue to achieve enhanced bioenergy anabolism by utilizing a synthetic electron shuttle and tuning inner nanostructures, holding great promise in wide-range ATP-powered bioapplications.

Valorization of castor oil-derived fatty acids for new bioactive compounds via chemo-enzymatic reactions

Novel lipidic compounds were prepared from 10-Undecenoic (UDA) and 11-bromoundecanoic acids (BUA) which are well known castor derived fatty acids. Ten novel structured monoacylglycerol (MAG)-based phenolic lipids were prepared from 11-bromoundecanoic acid by chemo-enzymatic approach and 20 new triazole based UDA hybrids were synthesized by Click chemical synthesis. The synthesized compounds were characterized by spectra analysis and studied for their antioxidant, antimicrobial and cytotoxic activities.

DNA flexible chain modified MOFs as a versatile platform for chemoenzymatic cascade reactions in glucose catalysis

Glucose oxidase (GOD) is widely used in the pharmaceutical industry, fermentation products and glucose biosensors for its essential role in catalyzing the conversion of glucose to gluconic acid and hydrogen peroxide (H2O2). As H2O2 is the by-product and will have a toxic effect on glucose oxidase, so introducing another enzyme that could consume H2O2 to form an enzymatic cascade reaction is a practical solution. However, this decision will lead to extra expenses and complex condition optimization such as the specific mass ratio, temperature and pH to improve the activity, stability and recyclability. Herein, we describe a mild and versatile strategy by anchoring GOD on carboxyl-activated MOF (Cu-TCPP(Fe)) through DNA-directed immobilization (DDI) technology. Robust MOF nanosheets were utilized as not only the carrier for the immobilization of GOD, but also a peroxidase-like catalyst for the decomposition of H2O2 to reduce its harmful impacts. In this work, the immobilized GOD retained 55.78% of its initial activity after being used for 7 times. More than 60% of the immobilized enzyme's catalytic activity was still maintained after 96 h of being stored at 50 ℃. This study provides a new idea for preparing immobilized enzymes with enhanced stability, fast diffusion and high activity, which can be used in fields such as biocatalysis and biotechnology.

Bottom-Up Synthesis of Multicompartmentalized Microreactors for Continuous Flow Catalysis.

The bottom-up assembly of biomimetic multicompartmentalized microreactors for use in continuous flow catalysis remains a grand challenge because of the structural instability or the absence of liquid microenvironments to host biocatalysts in the existing systems. Here, we address this challenge using a strategy that combines stepwise Pickering emulsification with interface-confined cross-linking. Our strategy allows for the fabrication of robust multicompartmentalized liquid-containing microreactors (MLMs), whose interior architectures can be exquisitely tuned in a bottom-up fashion. With this strategy, enzymes and metal catalysts can be separately confined in distinct subcompartments of MLMs for processing biocatalysis or chemo-enzymatic cascade reactions. As exemplified by the enzyme-catalyzed kinetic resolution of racemic alcohols, our systems exhibit a durability of 2000 h with 99% enantioselectivity. Another Pd-enzyme-cocatalyzed dynamic kinetic resolution of amines further demonstrates the versatility and long-term operational stability of our MLMs in continuous flow cascade catalysis. This study opens up a new way to design efficient biomimetic multicompartmental microreactors for practical applications.

Multi-compartmental MOF microreactors derived from Pickering double emulsions for chemo-enzymatic cascade catalysis

Bioinspired multi-compartment architectures are desired in synthetic biology and metabolic engineering, as credited by their cell-like structures and intrinsic ability of assembling catalytic species for spatiotemporal control over cascade reactions like in living systems. Herein, we describe a general Pickering double emulsion-directed interfacial synthesis method for the fabrication of multicompartmental MOF microreactors. This approach employs multiple liquid–liquid interfaces as a controllable platform for the self-completing growth of dense MOF layers, enabling the microreactor with tailor-made inner architectures and selective permeability. Importantly, simultaneous encapsulation of incompatible functionalities, including hydrophilic enzyme and hydrophobic molecular catalyst, can be realized in a single MOF microreactor for operating chemo-enzymatic cascade reactions. As exemplified by the Grubb’ catalyst/CALB lipase driven olefin metathesis/ transesterification cascade reaction and glucose oxidase (GOx)/Fe-porphyrin catalyzed oxidation reaction, the multicompartmental microreactor exhibits 2.24–5.81 folds enhancement in cascade reaction efficiency in comparison to the homogeneous counterparts or physical mixture of individual analogues, due to the restrained mutual inactivation and substrate channelling effects. Our study prompts further design of multicompartment systems and the development of artificial cells capable of complex cellular transformations.

Microperoxidase-11 functionalized nanozyme with enhanced peroxidase-mimicking activities for visual detection of cysteine

Various nanomaterials with peroxidase activity (nanozyme) have been designed for bio catalysis and biosensing, however, most of them need further design and modification of probe molecules for the specific binding reaction with targets. This results in a decrease in catalysis activity and hinders them to be perfect alternatives to natural enzyme in biosensing. In this work, an enhanced nanozyme was synthesized by functionalizing natural microperoxidase-11 (MP-11) on a hybrid graphene oxide-gold (GO-Au) material. The designed nanozyme showed an enhanced catalysis activity and realized a robust and efficient colorimetric detection of cysteine based on specific binding reaction between active iron center from MP-11 and thiol in cysteine. The enhanced properties show promising applications of complex nanozyme and provides a great opportunity for developing efficient sensing systems.

Integration of chemo- and bio-catalysis to intensify bioprocesses

Abstract Nature has evolved highly efficient and complex systems to perform cascade reactions by the elegant combination of desired enzymes, offering a strategy for achieving efficient bioprocess intensification. Chemoenzymatic cascade reactions (CECRs) merge the complementary strengths of chemo-catalysis and bio-catalysis, such as the wide reactivity of chemo-catalysts and the exquisite selective properties of biocatalysts, representing an important step toward emulating nature to construct artificial systems for achieving bioprocess intensification. However, the incompatibilities between the two catalytic disciplines make CECRs highly challenging. In recent years, great advances have been made to develop strategies for constructing CECRs. In this regard, this chapter introduces the general concepts and representative strategies, including temporal compartmentalization, spatial compartmentalization and chemo-bio nanoreactors. Particularly, we focus on what platform methods and technologies can be used, and how to implement these strategies. The future challenges and strategies in this burgeoning research area are also discussed.

New approaches to track chemoenzymatic loading reactions of NRPS carrier protein domains using NMR.

The atomic-level detail provided by nuclear magnetic resonance (NMR) allows for the monitoring of biochemical reactions as substrates and intermediates are converted by enzymes. NMR signals are sensitive to chemical environments and this feature allows for scrutiny of the integrity of reactions. Routinely, time-consuming diversions are necessary with traditional approaches to monitor biochemical transformations as many require additional sample manipulations or their readouts are limited. Here, we highlight the advantages of NMR to both monitor and rescue a complex chemoenzymatic reaction that modifies an isotopically labeled nonribosomal peptide synthetase (NRPS) carrier protein with an unlabeled phosphopantetheine moiety through an enzymatic cascade. We present protocols to monitor this reaction that implement various 1D NMR experiments tailored to the detection of specific steps in the enzymatic cascade and can be easily applied even to reactions using unlabeled domains. Additionally, to highlight the signals of the unlabeled modification once attached to the labeled biomolecule, we present a modified diffusion and isotope-filtered pulse sequence. Importantly, when chemoenzymatic reactions are slow or are stalled, the technique provides an atomic-level readout of reaction byproducts and limiting or damaged reagents, and thus allows for quick interventions to rescue them. The advantages of using NMR highlighted in this study more generally serve as a dogma to monitor and restore complex chemoenzymatic reactions.

Enzyme-metal-single-atom hybrid catalysts for one-pot chemoenzymatic reactions

Combining enzymatic and single-metal-atom catalysis is a promising approach for green chemical synthesis. We herein report a one-pot chemoenzymatic cascade reaction to asymmetric synthesize (R)-1-(4-biphenyl) ethanol by using the highly active and selective enzyme-metal-single-atom hybrid as the catalyst. We demonstrate that the Pd single atoms anchored lipase (Pd1/CALB-P) can efficiently drive one-pot cascade reactions in aqueous solution at 30 °C to achieve facile synthesis of chiral biaryl alcohols, which are important pharmaceutical intermediates that traditionally require complex synthesis procedures. The rate of (R)-1-(4-biphenyl) ethanol formation catalyzed by Pd1/CALB-P is more than 30-fold higher than that of the combination of commercial palladium on carbon (Pd/C) and lipase-pluronic conjugate (CALB-P). The enzyme-metal-single-atom hybrid catalyst provides a promising strategy for effectively merging the enzymatic and single-atom catalysis.

Chemo-enzymatic synthesis of sugar acid by pyranose 2-oxidase

Aldonic acids are valuable raw materials in the pharmaceutical, food, cosmetic, and construction industries. Herein, we report the biocatalytic synthesis of aldonic acids using pyranose 2-oxidase (P2O) to catalyze the key oxidation step of the sugar precursors. The reactions of P2O with monosaccharides in the presence of oxygen as an electron acceptor can result in the 2-keto, 2-keto sugar lactone, and 2-keto sugar acids, which can be further reduced to yield the corresponding aldonic acids. These chemo-enzymatic reactions can convert D-galactose and L-arabinose to galactonic acid and arabinoic acid, respectively, with a >99% conversion. We used methylation and high-resolution mass spectrometry to identify the product structures. Results of molecular docking and structural analysis explain how P2O controls the mechanism and regio-selectivity of the second oxidation of keto-sugars. The monosaccharides with OH at the C4 equatorial position favor C3 second oxidation, while the monosaccharides with C4-OH at the axial position favor C1 second oxidation. The data presented here explain for the first time at the molecular level how a sugar oxidase such as P2O produces two double oxidation products when different sugars are used as substrates.

Current Progress in the Chemoenzymatic Synthesis of Natural Products.

Natural products, with their array of structural complexity, diversity, and biological activity, have inspired generations of chemists and driven the advancement of techniques in their total syntheses. The field of natural product synthesis continuously evolves through the development of methodologies to improve stereoselectivity, yield, scalability, substrate scope, late-stage functionalization, and/or enable novel reactions. One of the more interesting and unique techniques to emerge in the last thirty years is the use of chemoenzymatic reactions in the synthesis of natural products. This review highlights some of the recent examples and progress in the chemoenzymatic synthesis of natural products from 2019–2022.

Enzyme-bimetallic hybrid catalyst for one-pot chemoenzymatic reactions

To overcome the intrinsic incompatibility between enzyme and chemical catalysts in one-pot chemoenzymatic cascade reactions, an enzyme-bimetallic hybrid catalyst was constructed by in situ reduction of ultrafine PdCu naonoclusters on the cross-linked lipase aggregates. The PdCu nanoalloy showed excellent activity in the Sonogashira coupling reaction because of the strong bimetallic synergistic effect between Pd and Cu within PdCu/CALB CLEAs. The activity of 3.4Pd1.7Cu/CALB CLEAs was 2.6-fold of Pd/CALB CLEAs in the synthesis of (R)-N-[1-(4-(phenylethynyl)phenyl)ethyl] acetamide. The highly active PdCu/CALB CLEAs rendered the one-pot chemoenzymatic reaction to perform at a lower temperature.

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins.

Post-translational glycosylation of proteins results in complex mixtures of heterogeneous protein glycoforms. Glycoproteins have many potential applications from fundamental studies of glycobiology to potential therapeutics, but generating homogeneous recombinant glycoproteins using chemical or chemoenzymatic reactions to mimic natural glycoproteins or creating homogeneous synthetic neoglycoproteins is a challenging synthetic task. In this work, we use a site-specific bioorthogonal approach to produce synthetic homogeneous glycoproteins. We develop a bifunctional, bioorthogonal linker that combines oxime ligation and strain-promoted azide–alkyne cycloaddition chemistry to functionalize reducing sugars and glycan derivatives for attachment to proteins. We demonstrate the utility of this minimal length linker by producing neoglycoprotein inhibitors of cholera toxin in which derivatives of the disaccharide lactose and GM1os pentasaccharide are attached to a nonbinding variant of the cholera toxin B-subunit that acts as a size- and valency-matched multivalent scaffold. The resulting neoglycoproteins decorated with GM1 ligands inhibit cholera toxin B-subunit adhesion with a picomolar IC50.

Recent applications of ionic liquid-based tags in glycoscience

The functionalization of glycosides with ionic compounds such as ionic liquids provides enhanced polarity for the labelled glycans thanks to the presence of a permanent positive charge. The chemical derivatisation of glycans with ionic liquids constitutes an emerging strategy to boost the detection sensitivity in MS applications. This allows the straightforward monitoring and detection of the presence of labelled glycans in complex matrices and in those cases where very limited amounts of material were available such as in biological samples and chemoenzymatic reactions. The use of ionic liquid based derivatisation agents can be further exploited for the labelling of live cells via metabolic oligosaccharide engineering for the detection of cancer biomarkers and for the tuning of live cells-surface properties with implications in cancer prognosis and progression. In this mini-review we summarise the latest development of the ionic liquid based derivatisation agents in glycoscience focussing on their use for sensitive MS applications.