Biocatalysis@TUDelft

Random copolymers containing charged, polar, and hydrophobic monomers protect enzymes so they can function in non-biological environments. These heteropolymers imitate unstructured proteins in coacervates that mimic membraneless organelles. Reporting in a recent issue of Nature, a team led by Xu and Alexander-Katz has designed heteropolymers with enzymatic activity by selecting monomers that modulate their chemical microenvironment to replicate enzymatic functionalities.

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

Unactivated alkenes rank among the most inert functional groups in synthesis and their selective reduction remains challenging. This Review charts the evolution from classical metal-catalyzed hydrogenation to radical hydrogen atom transfer (HAT) and emerging biocatalytic concepts, highlighting how complementary mechanistic strategies, including the recently introduced BioHAT, are reshaping how inert C═C bonds can be reduced.

ABSTRACT

The asymmetric reduction of unactivated alkenes remains a key challenge in synthesis due to their inherent lack of polarity and steric bias. Advances range from hydrogenation with molecular hydrogen and precious transition-metal catalysts, such as Crabtree's Ir and Noyori's Ru systems, to modern approaches employing abundant metals or radical-mediated hydrogen atom transfer (HAT) under mild conditions. Over the past decades, chemists have assembled a diverse mechanistic toolbox, with each strategy achieving excellent results under specific substrate, selectivity, and operating conditions. Clear trends have emerged toward greater sustainability, lower costs, and operational simplicity. Biocatalysis, previously limited to activated alkenes such as enones, has advanced via engineered promiscuous reductases, photobiocatalysis, and multifunctional enzymes, offering complementary and highly selective transformations. Enzymatic strategies for the reduction of unactivated alkenes, however, are still rare and highly substrate-specific. Recent innovations, such as BioHAT integrate radical-based mechanisms into engineered proteins and represent a potential first step toward general and practical biocatalytic solutions. The development of asymmetric reduction of unactivated alkene and synergistic integration of chemical and enzymatic strategies are summarized.

Mechanistic (experimental and theoretical) and structure–activity relationship studies of isocyanate generation from α-amino hydroxamic acid and sulfonyl fluoride under physiological conditions are reported. The optimized reaction offered a milder and more efficient affinity labeling technology than the gold standard photoaffinity labeling.

Target identification of bioactive compounds is of significance in life sciences, ranging from molecular biology to drug development. Photoaffinity labeling (PAL), which utilizes ultraviolet (UV) light irradiation to generate a reactive species for covalent bond formation, is the gold standard method for labeling the binding target. However, requirements for UV light irradiation, which can potentially cause denaturation of biomolecules, and uncontrollable reactivity, resulting in nonproductive consumption of the active species, necessitate further improvement of the affinity labeling methodology. Here, we report our studies on the in situ generation of isocyanate from an α-amino hydroxamic acid and a sulfonyl fluoride for affinity labeling. Theoretical and experimental mechanistic studies of the reaction using various α-amino hydroxamic acid derivatives provided a design principle for efficient isocyanate formation. The best α-amino hydroxamic acid showed higher covalent bond-forming efficiency than PAL in model protein modifications.

This review examines the structural and enzymatic properties of cyclic GMP-AMP synthase, including substrate promiscuity, homologs, and engineered variants. In addition, it evaluates potential expression systems for large-scale enzyme production and analyzes reaction and purification strategies for the synthesis of cyclic dinucleotides to connect fundamental enzymology to bioprocess development for pharmaceutical manufacturing.

Since its discovery as a pivotal enzyme in innate immunity, cyclic GMP-AMP synthase (cGAS) has been extensively studied for its immunological significance and catalytic mechanism. However, its potential as a biocatalyst for the efficient synthesis of the second messenger 2′3′-cyclic GMP-AMP (2′3′-cGAMP) remains underexplored. This review provides a comprehensive biotechnological perspective on cGAS, highlighting its enzymatic and structural features, substrate promiscuity, homologs, and engineered variants. We examined the expression systems reported in previous studies and assessed their suitability for scalable cGAS production. Furthermore, we explored reaction engineering strategies for 2′3′-cGAMP synthesis by comparing published production and purification methods. This review aims to bridge the gap between fundamental enzymology and applied bioprocessing by positioning cGAS as a promising biocatalyst for the pharmaceutical industry, with potential applications in immunotherapy, vaccine adjuvants, and beyond.

We report a sustainable enzymatic synthesis of caffeic acid phenethyl ester (CAPE) using Novozym 435 and bio-based solvents. A systematic approach combining solvent selection, design of experiments optimization, and mass transfer analysis achieved 75.6% yield under green conditions, highlighting the potential of biocatalysis for pharmaceutical applications.

Biocatalysis offers a sustainable alternative for chemical synthesis, but some enzymes, like lipases, still require conventional organic solvents, which are often flammable, toxic, and unsuitable for food or pharmaceutical applications. In this study, we present a systematic approach consisting of solvent selection, design of experiments optimization, and mass transfer analysis. As a case study, caffeic acid phenethyl ester (CAPE), a pharmacologically active compound derived from propolis, can be synthesized enzymatically. In this study, the Novozym 435-catalyzed esterification of caffeic acid and phenethyl alcohol was optimized using p-cymene, a bio-based solvent. To increase productivity, a system combining p-cymene as a solvent and dimethyl sulfoxide (DMSO) as a cosolvent was chosen. The optimal synthesis conditions were found to be 27 mM caffeic acid, 1460.5 mM phenethyl alcohol, and 73°C, achieving a 75.57% CAPE yield. Both external and internal mass transfer effects on the reaction rate were assessed. This study demonstrates the potential of using biocatalysts and green solvents for the sustainable synthesis of CAPE.

The synthesis of aromatic amino alcohols via a combination of an unspecific peroxygenase-catalysed reaction, oxyfunctionalization, and addition of selected nucleophiles and electrophiles in a highly atom-efficient manner was investigated.

This study presents the design to aim for an atom-efficient chemo-enzymatic synthesis route towards aromatic amino alcohols, based on an unspecific peroxygenase-catalysed oxyfunktionalisation of styrene and a highly atom-efficient conversion of the resulting epoxide with nucleophiles and electrophiles, respectively. This synthesis strategy features a simple two-step approach, and the practicality has been demonstrated at a semi-preparative scale. In a first step, the unspecific peroxygenase oxyfunctionalises the substrate, forming an epoxide. Due to its properties, the latter can serve as a starting material for the conversion into a wide range of products, thereby enabling the production of amino alcohols that are otherwise often difficult to synthesise. The shown concept features a one-pot two-step approach, depending on the respective ring-opening reagent. This method aims for a direct synthesis route for the pharmaceutical industry with good yields and high atom efficiency.

Proceedings of the National Academy of Sciences, Volume 123, Issue 8, February 2026.
SignificanceThis paper presents Pichia–Codon language model (Pichia-CLM), a deep learning–based language model for codon optimization to enhance recombinant protein production in the industrially relevant host,Komagataella phaffii. Unlike conventional ...

Amide formation represents a key reaction in pharmaceutical synthesis, for which more environmentally friendly methods are sought. In Science, Gao et al. report a biocatalytic oxidative strategy exploiting a redesigned aldehyde dehydrogenase that allows the coupling of aldehydes with amines at the expense of an oxidant [NAD(P)+, molecular oxygen].

Enzymes used on industrial scale are routinely engineered for best performance. However, exhaustive mutagenesis campaigns using the twenty canonical proteinogenic amino acids rapidly reach an evolutionary ceiling, where gains in activity compromise other critical properties such as thermal endurance. Although non-canonical amino acids (ncAA) expand the chemical space, most are costly for use on an industrial scale and significantly perturb structure. Here, we demonstrate that the evolutionary ceiling of highly optimized polyethylene terephthalate (PET) hydrolases (PETases) can be broken with azatryptophans that (i) differ minimally from their canonical tryptophan, (ii) are genetically encoded, and (iii) are produced in high yield by enzymatic biosynthesis from inexpensive precursors. The first genetic encoding systems are described for 4-azatryptophan, 5-azatryptophan, and 6-azatryptophan, achieving single, site-selective isosteric CH [->] N substitutions that enhancing the catalytic activity while preserving thermal stability. The fluorescence of 6AW provides a uniquely sensitive reporter of side-chain solvent exposure, which is critical for PETase activity and shown to vary between five different PETases. Furthermore, Azatryptophan-bearing enzymes are inexpensive to produce. To benchmark PETase activity, a rapid fluorescence-based kinetic assay, PETra, is introduced, which delivers consistency and reproducibility by using a soluble substrate yet correlates strongly with the hydrolysis of solid PET.

We examine how salt affects each step of the catalytic cycle of a halophilic glucose-6-phosphate dehydrogenase (G6PDH), which also catalyzes glucose oxidation (glcDH). We found that an increase in substrate binding rates is the main driving factor behind the increase in G6PDH activity, whereas the acceleration of the chemical step underlies the increase in glcDH activity. The role of charge screening as a key factor is discussed.

Enzymes from halophilic organisms function in near-saturating salt concentrations. To explore enzyme adaptation to these conditions and the broader effects of salt on enzyme catalysis, we examined the salt effect on the kinetics of a glucose-6-phosphate dehydrogenase from the halophilic archaeon Haloferax volcanii (HvG6PDH). This enzyme has a negatively charged surface and catalyzes the oxidation of the negatively charged substrate glucose-6-phosphate (G6PDH activity) and glucose (glcDH activity). We found strikingly different salt effects on the two activities. Steady-state kinetics experiments revealed a 50-fold decrease in K _M ^G6P for G6PDH activity. Instead, for glcDH activity, the main effects were a 10-fold increase in k _cat and a 10-fold increase in k _cat ^glc/K _M ^glc. The effect of salt in each step of the catalytic cycle was assessed using pre-steady-state kinetic experiments. We found that KCl increases both the binding and release rates of NAD⁺ in G6PDH activity, whereas in glcDH it does not affect binding but decreases the release rate. In G6PDH, KCl does not affect the chemical step, while in glcDH this step is significantly accelerated. The results further indicate that the increase in G6P affinity is the main contributor to the increase in G6PDH activity, whereas the increase in glcDH activity is primarily driven by the increase in the chemical step rate. Together, these findings highlight charge screening as a key factor underlying the differential effects of salt, by reducing electrostatic repulsion between the enzyme surface and its substrates and modulating interactions between the positively charged active site and the substrates.

Here, we summarise the structural features of BcABA3 and its homologues. ABA3 proteins adopt an all-α-helix fold and utilise a distinct substrate-binding mode to catalyse FPP cyclisation. These enzymes contain a Mg²⁺ ion-coordinating Glu, a PPi-interacting RY pair, a Zn²⁺ ion-binding motif, and a KLW motif-harbouring lid that seals the catalytic centre, providing a valuable foundation for further investigations into this new class of terpenoid cyclases.

ABA3 proteins are a group of terpenoid cyclases (TCs), which share very low sequence identity with known proteins and lack signature Mg²⁺ ion-binding motifs used to define TCs. The first characterised ABA3 from Botrytis cinerea catalyses the cyclisation of farnesyl pyrophosphate (FPP) via an unconventional process to produce (2Z,4E)-α-ionylideneethane, the skeleton of one of the major phytohormones—abscisic acid. We recently resolved the crystal structures of BcABA3 and two closely related proteins. BcABA3 and RuABA3 from fungi are (2Z,4E)-α-ionylideneethane synthases, whereas SkABA3 from a bacterium transforms FPP to sesquiterpene, whose identity remains unelucidated. ABA3 proteins adopt an all-α-helix fold that contains a Zn²⁺ ion-binding motif, and exploit unique features to coordinate Mg²⁺ ions and bind pyrophosphate of the substrate. In addition, the lid region undergoes extensive conformational change upon the binding of the substrate, and thoroughly covers the catalytic centre. The identification of ABA3 proteins is an important advance for natural product research, organic chemistry and enzymology, and the structural information summarised here should be important to guide in-depth investigations of this class of TCs.

Nature Chemistry, Published online: 25 February 2026; doi:10.1038/s41557-026-02079-9

The enzymatic synthesis of azetidines is a prime example of the superiority natural systems often show over laboratory syntheses, but how nature achieves such difficult transformations in mild conditions is unclear. Now, two independent reports have revealed that the azetidine ring of polyoxamic acid arises from L-isoleucine via a dual-enzyme system that overcomes major energetic barriers through coordinated metalloenzyme chemistry.

A tetrazine-mediated inverse electron-demand Diels–Alder (IEDDA) reaction enables selective modification of allyl-functionalized RNA. Unstrained N ⁶- and 2′-O-allyl adenosines serve as reactive handles for cycloaddition under controlled conditions, defining a chemical framework for RNA functionalization and expanding the scope of IEDDA chemistry beyond strained alkene systems.

The inverse electron-demand Diels–Alder (IEDDA) reaction has emerged as a powerful tool for biomolecular conjugation, yet its application to nucleic acids has been largely limited to strained or synthetically demanding dienophiles. Here, we report a general and bioorthogonal strategy for RNA labeling by leveraging enzyme-installable allyl modifications as dienophile handles for tetrazine cycloaddition. Guided by frontier-orbital analysis, we identified 1,2,4,5-tetrazine-3,6-dicarboxylate (Tz 5) as an activated electron-deficient diene capable of engaging electronically unactivated allyl handles in IEDDA reactions under nonaqueous conditions. Tz 5 reacts efficiently with both N6-allyladenosine (a⁶A) and 2′-O-allyladenosine (A_a), forming stable cycloaddition adducts in nucleosides and within RNA oligonucleotides without perturbing canonical bases or phosphate linkages. The IEDDA labeling proceeds with high selectivity, and enzymatic digestion confirms site-specific conjugation exclusively at the allyl-modified nucleosides. These findings expand the scope of IEDDA chemistry to unstrained, electron-rich terminal alkenes in RNA and establish a⁶A and A_a as versatile, orthogonal handles for tetrazine-mediated labeling. This article offers a broadly applicable platform for selective RNA functionalization and provides new opportunities for probing, imaging, and manipulating RNA in complex biological environments.

Adipic acid (1,6-hexanedioic acid) is a key building block for nylon-6,6, a widely used polymer in the global chemical industry. Current industrial production relies on petrochemical feedstocks and nitric acid oxidation of cyclohexane/cyclohexanol mixtures, releasing nitrous oxide, a potent greenhouse gas. Biotechnological routes offer sustainable alternatives but have been limited by low yields or reliance on multi-strain systems. Here we report a one-pot, single-strain microbial process for the efficient conversion of guaiacol - a lignin derived aromatic - into adipic acid. By integrating heterologous Rieske non-heme iron monooxygenases from Cupriavidus necator N-1 with systematic process optimisations in engineered Escherichia coli, we achieve near-quantitative conversion with 97% yield and titres of 1.5 g/L in aqueous, lab-scale reactions. This work demonstrates a novel and efficient strategy for lignin valorisation through engineered microbial synthesis, providing a new sustainable and scalable route to adipic acid.

To overcome the limitation of toxic byproduct generation in NO removal, this work presents a biohybrid strategy that leverages natural enzymatic active sites to enhance exciton dissociation, thereby achieving efficient deep NO oxidation. By immobilizing cytochrome c on carbon nitride, the semi-artificial photoenzyme exhibits powerful internal electric field with a 2.4-fold increase in surface potential, enabling efficient deep NO oxidation.

ABSTRACT

Solar-driven catalysis holds promise in molecular oxygen activation and low-concentration NO_x elimination but achieving highly selective conversion of NO to nontoxic nitrate (NO₃ ⁻), while suppressing toxic NO₂ formation remains challenging. Here, we report a semi-artificial photoenzyme catalyst constructed by immobilizing mono-heme cytochrome c (cyt c) on carbon nitride (CN) to refine reactive species generation for deep NO oxidation. The hybrid photoenzyme catalyst establishes localized internal electric fields (IEF) between cyt c and CN that promote exciton dissociation, and creates polarized active sites on cyt c that preferentially adsorb O₂, and activate O₂ to active peroxides (•O₂ ⁻ and *O₂ ²⁻) via an electron transfer pathway with inhibiting ¹O₂ formation. The one-step NO oxidation to NO₃ ⁻ is boosted over cyt c/CN, showing exceptional 97.4% NO conversion with ultralow NO₂ selectivity (1.5%, 6.5 ppb) and long-term stability (98.1% after 300 min) at a weight hourly space velocity (WHSV) of 850 L g⁻¹·h⁻¹ across wide humidity ranges. The undesirable NO₂ generation is significantly lower than that of bare CN (17.0%, 104.7 ppb) and reported catalysts. The catalyst exhibits high activity in direct NO₂ removal (92.9%) and rapidly reduces NO levels to below the safe concentration (52 ppb) in a simulated environment chamber.

Nature Communications, Published online: 23 February 2026; doi:10.1038/s41467-026-69968-y

L-Proline is a powerful organocatalyst widely applied in asymmetric synthesis due to its secondary amine functionality, however, in proteins, this functional group is locked in peptide bonds, rendering proline catalytically inactive. Here, the authors engineer the nonenzymatic protein scaffold LmrR into a new-tonature biocatalyst by exposing its native L-proline residue at the N-terminus to catalyze enantioselective aldol reactions.

Nature Catalysis, Published online: 23 February 2026; doi:10.1038/s41929-026-01493-z

Despite its importance in medicinal chemistry, the sulfonamide functional group is rare in natural products and its biosynthesis is poorly understood. This study reveals the structure and mechanism of SbzM, a sulfonamide synthase essential for altemicidin biosynthesis.