Biocatalysis@TUDelft

Applied and Environmental Microbiology, Ahead of Print.

Applied and Environmental Microbiology, Ahead of Print.

Proceedings of the National Academy of Sciences, Volume 123, Issue 12, March 2026.
SignificanceWhile nature has evolved enzymes to carry out a vast array of chemical transformations, selecting the ideal protein to initiate an enzyme engineering campaign often presents a significant challenge, slowing progress across biocatalysis and ...

Nature Communications, Published online: 24 March 2026; doi:10.1038/s41467-026-70981-4

Halogenation enhances the stability and function of pharmaceuticals and biomaterials, however, current halogenase enzymes are inefficient and insoluble. Here the authors use continuous evolution to engineer a soluble and active halogenase that can produce 2.7 g/L of halogenated tryptophan.

Nature Communications, Published online: 24 March 2026; doi:10.1038/s41467-026-70996-x

Structural complexity often hinders the efficient conversion of lignin into sustainable high-value products. This bifunctional core–shell catalyst enables a relay reaction that transforms lignin into jet-fuel range cycloalkanes with high yields.

Nature Chemical Biology, Published online: 23 March 2026; doi:10.1038/s41589-026-02177-2

How microorganisms build the catalytic heart of the nitrogenase enzyme has remained unknown. Two studies now show how these enzymes repurpose a nitrogenase-like scaffold to assemble the nitrogenase cofactor.

Nature Chemical Biology, Published online: 23 March 2026; doi:10.1038/s41589-026-02179-0

The maturation of the unique FeMo-cofactor of molybdenum nitrogenase is a multistep process requiring the sequential action of a series of maturase complexes. Here, the authors report on how cryo-electron microscopy structures show NifB-co transfers from NifX to NifEN’s internal site, where NifB-co is converted into FeMo-co for insertion into Mo-nitrogenase.

Nature Synthesis, Published online: 24 March 2026; doi:10.1038/s44160-026-01043-2

Redesigning non-haem iron enzymes to incorporate a nickel centre enables ligand-to-metal charge transfer-driven photoenzymatic C(sp²)–S cross-coupling.

Nature Chemistry, Published online: 18 March 2026; doi:10.1038/s41557-026-02085-x

We developed a strategy to repurpose rare codons in mammalian cells, enabling the simultaneous incorporation of up to five distinct noncanonical amino acids into a single protein. By avoiding previous limitations in genetic code expansion using stop codons, this rare codon recoding facilitated advanced protein engineering applications.

Pickering droplet derived microreactors are engineered for the co-confinement of enzymes and cofactors, achieving strong retention, freely mutual accessibility, and in situ cofactor regeneration. These self-sufficient systems enable continuous flow catalysis with excellent enantioselective performance (>80% conversion, >99% ee), long-term operational stability (2 000 h), and high cofactor total turnover number (173 907 mol mol⁻¹).

ABSTRACT

The co-immobilization of enzymes and cofactors represents a sustainable platform for continuous-flow synthesis of chiral pharmaceuticals, yet balancing effective retention with mutual accessibility of them remains challenging. Herein, we report a sol-gel strategy to construct Pickering droplet derived microreactors (PDMRs) for the co-confinement of enzymes and cofactors, which have been applied to continuous flow reactions without exogenous addition of cofactors. Within these self-sufficient PDMRs, the cofactors are reversibly immobilized via electrostatic interactions, enabling in situ regeneration and free access to the enzyme. The PDMRs efficiently encapsulate enzymes and cofactors with 85–100% immobilization efficiency and robust thermal stability. In PDMRs-catalyzed continuous flow reactions, excellent catalytic performance and high cofactor total turnover number (TTN) were obtained in nicotinamide adenine dinucleotide phosphate (NADP⁺)-dependent aldo-keto reductase (AKR) catalyzed enantioselective reductions (80–100% conversions, >99% ee, 500 h stability, up to 173 907 mol mol⁻¹ TTN), and pyridoxal 5-phosphate (PLP)-dependent transaminase (TA) catalyzed enantioselective transaminations (80–100% conversions, >99% ee, 2 000 h stability, up to 45 552 mol mol⁻¹ TTN). Furthermore, the PDMRs are extended to the co-confinement of a multi-enzyme system (AKR and glucose dehydrogenase, GDH) with NADP⁺ for chiral alcohols synthesis with sustained operational stability. This work establishes a potent and durable strategy for industrial-scale continuous flow manufacturing.

The pyrrolysyl-tRNA synthetase dimer exhibits an alternating mode of action of its monomers within its catalytic cycle, which is realized upon occupation of secondary binding sites located at the intermonomer interfaces outside the catalytic binding site.

ABSTRACT

Aminoacyl-tRNA synthetases mediate the activation and transfer of amino acids to their cognate tRNA, which constitutes one of the initial events in protein biosynthesis. Even though different mechanisms of action have been proposed for the catalysis of these enzymes, their entire catalytic cycle remains elusive. Here, we used electron paramagnetic resonance spectroscopy in vitro and in cells in combination with molecular dynamics simulations to study the role of amino acid interactions in the catalytic cycle of pyrrolysyl-tRNA synthetases (PylRS), a widely used tool for genetic code expansion. Experiments using the paramagnetic non-canonical amino acid SLK-1 revealed the presence and occupation of secondary amino acid binding sites in PylRS located at the intermonomer interface, distant from the catalytic binding site. Based on our results, we propose a model that assumes an alternating mode of action of the two PylRS monomers for the catalytic cycle of PylRS.

Nature Synthesis, Published online: 23 March 2026; doi:10.1038/s44160-026-01014-7

The condensation domain PvdL-C3 is shown to synthesize the tetrahydropyrimidine ring of pyoverdine, revealing cyclization capability in a canonical non-ribosomal peptide synthetase domain. Structural and mutagenesis analyses identify key active site residues that control catalytic specificity and stereochemistry, providing a structural blueprint for enzyme engineering to produce bioactive peptides.

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Plant-type (Form I) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) suffers from inherent catalytic trade-offs and a strong dependency on other proteins--including an essential small subunit (SSU) and auxiliary chaperones--for assembly, constraining the enzymes evolutionary and engineering potential. Here, we investigated representatives from the newly discovered clade Form IF. These enzymes do not require specific chaperones to form functional complexes, exhibit high CO2-specificities (SC/O [~]50) while maintaining high turnover rates (up to kcat [~]11 s-1). Remarkably, two Form IF representatives (IF-1/IF-2) lost the dependency on the SSU and assemble into homo-octameric complexes without their cognate SSUs. While the SSU is not necessary for catalysis, its addition improves both activity and specificity in IF-1/IF-2. Our results show that complexity is actually not required to achieve highly active, specific and functional Rubisco variants--and that this complexity can even be reverted--which challenges our current thinking on the evolution and catalytic mechanism of Rubisco.

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@189e5ceorg.highwire.dtl.DTLVardef@4e4581org.highwire.dtl.DTLVardef@b556e2org.highwire.dtl.DTLVardef@15b244f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Optimizing BHET product levels by ethylene glycol content during enzymatic degradation of Poly(Ethylene Terephthalate) with the enzyme LCC_ICCG.

Recycling of enzymatically depolymerized poly(ethylene terephthalate) (PET) involves polycondensation of bis(2-hydroxy-ethyl) terephthalic acid (BHET)—a degradation product of enzymatic PET hydrolysis. The recycling process is simplified when more BHET is generated by the enzymatic reaction. Here, we report how ethylene glycol (EG) addition can maximize BHET formation using leading PET hydrolases, LCC_ICCG, and PHL7. EG at any level above 2–5% vol/vol was found to decrease the steady-state enzymatic degradation rates while enhancing the relative production of BHET. For LCC_ICCG, the highest measured BHET levels (product fraction approaching 0.5) were attained at EG levels of ∼27–29% and reaction temperature ∼62.5°C. EG shortened the enzymatic reaction lag-phase and lowered the lag-phase increase with PET crystallinity. EG works by perturbing the adsorption, including nonproductive adsorption, of the enzymes to the PET surface, which manifests as an apparent change in substrate affinity (increases the ^inv K _m in interfacial kinetics modeling) and directs the enzyme more to the liquid phase.

We report a photoenzymatic hydroalkylation that enables streamlined, stereocontrolled access to aryl glutarimide precursors relevant to targeted protein degradation. Engineered flavin-dependent “ene”-reductases provide broad scope and high enantioselectivity through a distinct electron transfer–enantioselective proton transfer pathway.

ABSTRACT

We describe a photoenzymatic hydroalkylation reaction that enables the efficient and stereocontrolled synthesis of aryl glutarimide precursors—chemically and configurationally robust entry points to bioactive agents for targeted protein degradation. Screening of flavin-dependent “ene”-reductases identified GluER HA _rac , a G. oxydans variant, as an efficient and substrate-tolerant catalyst, granting access to >30 (hetero)aryl glutarimide precursors. A directed evolution campaign then furnished a hexamutant, GluER HA _ent , that delivers the products in up to 93:7 enantiomeric ratio. Mechanistic experiments revealed a pathway that departs from the hydrogen atom transfer mechanism previously established for related systems, proceeding instead via radical–polar crossover followed by enantioselective proton transfer from an active-site tyrosine residue. Collectively, these studies establish a biocatalytic platform for advancing the synthesis and diversification of glutarimide-containing degraders.

Protein macrocyclization was applied to a thermally unstable D-stereospecific hydrolase using in situ cyclization of proteins (INCYPRO). This site-specific cross-linking approach enhanced the resistance of the enzyme to heat and cosolvents. A cross-linked dimer with improved activity and stability was identified and structurally confirmed, demonstrating the effectiveness of macrocyclization for protein engineering.

ABSTRACT

Enzymes are powerful catalysts for selective transformations but often suffer from limited stability under operational conditions such as elevated temperature or the presence of organic cosolvents. While sequence-based strategies have been widely used to improve stability, chemical protein engineering enables modifications beyond the natural amino acid repertoire thereby offering complementary routes to tailor enzyme function and robustness. Here, we apply the in situ cyclization of proteins (INCYPRO) to a D-stereospecific hydrolase with low intrinsic thermal stability. Site-specific macrocyclization substantially improved resilience to heat and cosolvent stress. Unexpectedly, we discovered a cross-linked protein dimer with enhanced activity and thermal stability. The complex structure was confirmed by x-ray crystallography. Extending the INCYPRO approach, we engineered a multicyclic enzyme dimer with a total of four cross-linking sites, which not only retained high activity under benign conditions but also outperformed the wild-type under stress. Our findings establish protein macrocyclization as a versatile strategy to stabilize both monomeric and multimeric enzymes, providing a powerful route to robust biocatalysts.