Biocatalysis@TUDelft

Applied and Environmental Microbiology, Ahead of Print.

Applied and Environmental Microbiology, Ahead of Print.

Accelerating the development of enzymatic degradation of polyesters such as poly(ethylene terephthalate) (PET) and poly(butylene terephthalate) (PBT) requires a rapid and parallelizable detection method. We developed a protein-based biosensor for the fast and accurate quantification of the PET and PBT degradation product, terephthalate (TPA), which we named TPAsense. Engineering TPAsense required overcoming low thermal stability and aggregation of the initial construct by introducing stabilizing mutations without disrupting the binding affinity to TPA. The sensor performance was validated by screening for the PBT degrading activity of a Leaf-branch Compost Cutinase (LCC) mutant library and comparing with liquid chromatography data. TPAsense detects nanomolar concentrations of TPA enabling shorter incubation times for screening workflows. In addition, a comparative analysis of PETase and PBTase kinetics was performed with TPAsense. Finally, we demonstrated the detection of PET microplastic in samples from a wastewater treatment plant by combining the biosensor and a PETase. TPAsense offers a platform to accelerate PETase and PBTase development for plastic waste recycling and detection of microplastic in the environment.

Adenine is a ubiquitous nucleobase found in nucleic acids, cofactors, and signaling molecules and mediates diverse molecular interactions. Here, we identify TvAPT, an adenine prenyltransferase from the cyanobacterium Trichormus variabilis NIES-23. Unlike canonical enzymes limited to C5 dimethylallylation, TvAPT efficiently catalyzes the unprecedented N6-prenylation of adenine-containing substrates using extended prenyl donors (C10 and C15), markedly increasing the hydrophobicity of the adenine moiety. X-ray structural analysis and protein engineering revealed that an enlarged prenyl-binding pocket enables this donor promiscuity, allowing rational tuning of prenyl-donor preference. These findings establish TvAPT as a versatile biocatalytic platform that expands the chemical space of adenine-containing molecules for biomolecular engineering, as demonstrated by the synthesis of membrane-permeable nucleotides and analogues of plant signaling molecules.

Cofactor engineering is revolutionizing green biomanufacturing by overcoming the fundamental bottleneck between the limited supply/regeneration of cellular energy cofactors [e.g., NAD(P)H, ATP] and the high demands of efficient bioproduction. This review highlights advanced strategies, such as orthogonal systems that separate product synthesis pathways from basal metabolism, external energy sources (e.g., light or electricity) for cofactor regeneration, and material-enabled immobilization for scalable processes. These approaches enable high-yield production of diverse compounds, from specialized optically pure pharmaceuticals to bulk chemicals, by addressing critical limitations in yield, purity, and industrial scalability beyond conventional fermentation. Finally, we discuss challenges in process stability and economic viability, underscoring cofactor engineering’s potential as a versatile strategy for sustainable, next-generation biomanufacturing.

Nature Catalysis, Published online: 02 April 2026; doi:10.1038/s41929-026-01515-w

Radical C(sp3)–C(sp3) bond formation with stereocontrol is challenging. Now, photoredox catalysis and repurposed thiamine-dependent enzymes are combined to couple cinnamyl aldehydes with benzylic radicals, yielding enantioenriched carboxylic acids bearing one or even two stereocentres.

We present a versatile approach to uniform hybrid microgels containing magnetite nanoparticles (MNPs). Methylcellulose stabilizes MNPs in pre-hydrogel suspensions, enabling microfluidic processing. Functionalization with biotin yields an experimental platform for enzyme immobilization, shown by immobilizing streptavidin-horseradish peroxidase (HRP) and a colorimetric volume activity analysis using a 3,3',5,5'-tetramethylbenzidine (TMB) oxidation assay. The microgels' magnetic properties enable simple recovery of bound enzymes.

We introduce droplet microfluidics-fabricated hybrid microgels composed of biotinylated acrylamide that is crosslinked with N,N’-methylenebis(acrylamide). These microgels are further functionalized with streptavidin and loaded with magnetite nanoparticles (MNPs). During microfluidic processing, MNPs remain dispersed in an aqueous methylcellulose solution for over 4 h due to increased viscous drag, enabling stable droplet formation. In contrast, the MNPs sediment within a few minutes in pure aqueous solution. The resulting multifunctional hybrid microgels facilitate straightforward protein immobilization under mild conditions, for example, utilizing enzymes conjugated with streptavidin or recombinant fusion proteins of magnetite-binding proteins. Simultaneously, our microgels enable the recovery of immobilized proteins through magnetic separation of the microgels from solution. The quantity of immobilized proteins can be regulated independently by varying the amount of coupled biotin or encapsulated MNPs. Using microgels containing different quantities of coupled biotin, we demonstrate binding of a streptavidin-conjugated fluorescent dye and horseradish peroxidase. To confirm the availability of MNPs for magnetite-binding proteins, a fusion protein of the magnetite-binding protein Mad10 and super-folder green fluorescent protein (sfGFP) was immobilized, and its fluorescence was detected.

Haloalkane dehalogenases catalyze an SN2 nucleophilic substitution to erase halogen substituents in organohalogen compounds. The acid-base-nucleophile triad secures irreversible SN2 displacement of the halogen for the hydroxyl derived from the water. In this report, we elucidate a molecular basis for how this originally irreversible SN2 process becomes fully reversible in a histidine-to-phenylalanine mutant, favoring a transhalogenation chemistry. This offers a greener alternative for industrial transhalogenation processes, applicable in the recycling of environmentally harmful halogenated compounds.

Directed versus Innate: Precision Oxygenation at the Endgame – Late-stage oxygenation (LSO) enables the selective installation of oxygen functionality in complex molecules at advanced stages of synthesis. Increasingly important in drug discovery, agrochemicals, and materials science, modern LSO strategies integrate chemical, photochemical, electrochemical, and enzymatic oxidation methods. This Review highlights key mechanistic concepts, reactivity patterns, and emerging strategies for predictable oxygenation in complex settings.

ABSTRACT

Late-stage oxygenation (LSO) has emerged as a transformative strategy in modern synthetic chemistry, enabling the precise modification of complex, densely functionalized molecules. Driven by the growing demand for efficient, selective, and sustainable methods in pharmaceutical, agrochemical, and materials science, recent advances have expanded the scope and utility of this approach. Breakthroughs in the design of selective oxidants, biocatalytic platforms, and photoredox methodologies have redefined the possibilities for molecular diversification and functionalization at advanced synthetic stages. This review consolidates the conceptual foundations of LSO with cutting-edge developments across chemical and enzymatic domains, highlighting both practical applications and mechanistic insights. By integrating these perspectives, we aim to provide a comprehensive resource that will guide practitioners across disciplines, from those newly entering the field to experts seeking to harness the latest innovations.

A motif-based deep learning method is developed to overcome the limitations of traditional sequence alignment, enabling the discovery of 95 previously unknown NADH-dependent imine reductases. This work not only expands the imine reductase toolbox for amine synthesis but also establishes a generalizable strategy for enzyme discovery.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@10d34f1org.highwire.dtl.DTLVardef@ec3fcorg.highwire.dtl.DTLVardef@164fa59org.highwire.dtl.DTLVardef@700a99_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nature Chemistry, Published online: 30 March 2026; doi:10.1038/s41557-026-02106-9

Selective activation of aliphatic C–H bonds in complex polycyclic terpenoids remains a significant challenge. Now a bacterial P450 enzyme has been shown to achieve precise functionalization of inert C–H sites in pentacyclic triterpenoids, unveiling a distinctive relay-oxidation pathway mediated by 1,5-hydogen atom transfer and establishing a versatile chemo-enzymatic platform for accessing previously unexplored chemical space.

Nature Chemical Biology, Published online: 25 March 2026; doi:10.1038/s41589-026-02169-2

Song et al. find that biomolecular condensates can catalyze reductive amination of metabolites through a nonenzymatic mechanism, mediating C–N bond formation in vitro and impacting cellular metabolism in Escherichia coli.

Nature Catalysis, Published online: 26 March 2026; doi:10.1038/s41929-026-01498-8

Biological nitrogen fixation is vital for sustainable agriculture, yet nitrogenase engineering is hindered by limited insight into their essential metallocluster cofactor assembly and transfer. Here, by capturing the nitrogenase biosynthetic component NifEN in multiple structural states, a tunnel-and-switch mechanism that coordinates receipt, maturation and delivery of the FeMo-cofactor precursor is revealed.

This work presents a convergent, stereodivergent total synthesis of glabridin. A lipase-mediated DKR, combined with protecting-group engineering and fragment coupling, enables access to both enantiomers in short sequences and offers a general blueprint for the synthesis of isoflavan and related polyphenolic natural products.

ABSTRACT

Herein, we describe a modular, stereodivergent chemoenzymatic strategy for the enantioselective total synthesis of the natural products (+)- and (–)-glabridin. A lipase-catalyzed dynamic kinetic resolution establishes the key benzylic stereocenter, while a carefully engineered protecting-group manifold preserves stereochemical integrity during fragment coupling and cyclization. From inexpensive, commercially available resorcinol-derived building blocks, the sequences deliver (–)-glabridin in 10 steps with 14% overall yield and (+)-glabridin in 12 steps with 7% overall yield. This convergent platform provides practical access to both enantiomers of glabridin and offers a general blueprint for the stereocontrolled synthesis of structurally related polyphenolic natural products.

A novel quercetin 2,3-dioxygenase was engineered to reshape its substrate-binding cavity and redirect its specificity toward artificial flavonols. One variant with a larger substrate-binding cavity exhibited 20- to 1750-fold higher activity toward bulky flavonols with phenyl-based substitutions at position C-8. In contrast, a variant with a smaller substrate-binding cavity showed 15-fold higher activity toward the smaller flavonol 3,7-dihydroxyflavone.

ABSTRACT

A novel quercetin 2,3-dioxygenase from Penicillium chrysogenum, following biochemical characterization, served as the starting point for reshaping the substrate-binding cavity to alter its substrate specificity. Using a rational engineering strategy supported by computational predictive tools, we achieved high activity toward specific artificial flavonols. In all generated variants, amino acids were replaced with residues that naturally occur at the selected positions in homologous enzymes. Two variants with enlarged substrate-binding cavities exhibited improved activity toward bulkier substrates. In particular, the Y55F-F134L-M143L variant showed 20- to 1750-fold higher activity toward flavonol compounds with phenyl-based substitutions at position C-8. Conversely, one variant with a smaller substrate-binding cavity showed 15-fold higher activity toward the smaller flavonol 3,7-dihydroxyflavone. The procedure described here has implications for engineering metalloenzymes to alter their substrate specificity toward novel compounds.