Biocatalysis@TUDelft

Proceedings of the National Academy of Sciences, Volume 122, Issue 26, July 2025.
SignificanceTerpenoid natural products have featured prominently in the pharmacopoeia since times of antiquity. For example, C15sesquiterpenes are responsible for the analgesic effects of myrrh, historically prescribed as a painkiller in the Ebers ...

Biocatalysis offers an attractive route in the development of more sustainable approaches in silicon chemistry. Given the propensity for enzymes to operate optimally in an aqueous environment there are obvious challenges to incorporating biocatalysts in organic solvents. The modification of proteins with siloxanes represents a unique opportunity to perform homogeneous biocatalysis in organic solvents without negatively impacting enzyme activity.

Abstract

Biocatalysis presents an interesting opportunity for addressing the need for sustainability in chemical processes, especially as a means of supplanting catalysts based on nonrenewable metals. However, a significant challenge facing this strategy is the propensity for biological molecules to function optimally in aqueous environments while many chemical transformations occur in organic solvents, an environment that is typically antithetical to the functioning of enzymes. To address this challenge, we have modified proteins with siloxane oligomers in an effort to generate biocatalytic systems that can be used in homogeneous reaction systems rather than as heterogeneous catalysts where the biocatalyst is immobilized on a solid support. The siloxane-modified proteins displayed activity and stability in organic solvents that is comparable to that observed with unmodified proteins in aqueous environments and demonstrated excellent solubility in organic solvents. Modification of the proteins was a straightforward process that demonstrated a high level of efficiency. The covalent modification of human serum albumin (HSA) and trypsin with siloxanes was examined using matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF-MS) and the Michaelis-Menton activity of the enzyme was studied using standard assays.

Anchoring an enzyme at oil/water interface was established by conjugating it with an amphiphilic polymer. This interfacial anchoring facilitated substrate acquisition and enzyme conformation dynamics conductive to reaction kinetics, thereby expanding the application scope of enzymatic catalysis, as demonstrated through in situ absorption and hydrolysis of 1-bromobutane.

Abstract

The enzymatic degradation of volatile haloalkane waste gases, which are commonly generated from halogenation reactions in organic chemical synthesis, is challenging due to their low concentration and poor aqueous solubility. In this study, we proposed a novel process that integrated in situ substrate absorption using an organic solvent with enzymatic degradation, using haloalkane dehalogenase LinB as a model enzyme, 1-bromobutane as a volatile haloalkane, and n-dodecane as the absorbent. An amphiphilic polymer, Pluronic F127, was grafted onto LinB, resulting in the Pluronic-LinB conjugate being anchored at the n-dodecane/water interface, as evidenced by confocal laser scanning microscopy. The interfacial anchoring facilitated the uptake of 1-bromobutane by LinB, thereby enhancing catalytic hydrolysis. Pluronic-LinB exhibited a significantly enhanced flexibility at the n-dodecane/water interface, as shown by low-field nuclear magnetic resonance. All these increased enzymatic performances, as could be interpreted from the kinetic parameters. Moreover, molecular dynamic simulations revealed that the attached polymer enhanced the conformational dynamics of helix and loop motifs forming the substrate channels of the enzyme, increased the water flux, and reduced the residence time of water molecules in the active pocket. This work has presented a novel molecular engineering strategy to expand the application spectrum of enzymatic catalysis.

A dual-functional catalyst that unites peptide catalysis and biocatalysis within a single protein scaffold at two distinct active sites offers a promising strategy for cascade catalysis. This hybrid catalyst enabled here a one-pot transformation of alcohols to C─C coupled products via enamine activation, revealing challenges such as mismatched reaction rates and interference by lysine residues.

Abstract

A single protein that catalyzes two different reactions at two distinct active sites within one structural domain may be beneficial for cascade reactions. We explored this approach by merging a tripeptide catalyst with an alcohol dehydrogenase as a case study to bring together the strengths of enzymatic redox chemistry and peptide-catalyzed carbon–carbon bond formation. A proline-based peptide catalyst for C─C bond formation relying on an enamine intermediate was successfully merged to the N-terminus of an alcohol dehydrogenase to catalyze a cascade involving alcohol oxidation followed by C─C bond formation. It turned out that the reaction speed of the two catalytic sites diverged, and the ε-amino group of lysine residues present in the enzyme interfered with the organocatalytic proline activity. Removing/exchanging lysine residues within the enzyme reduced the background reaction but also the native redox activity. Interestingly, exchanging the N-terminal proline with a histidine switched the stereopreference. The simultaneous cascade reaction of alcohol oxidation to the aldehyde and C─C bond formation with nitrostyrene presents a first proof-of-concept for bringing peptide catalysis together with enzyme catalysis and creating a bi-active site catalyst within a single domain.

Immobilized enzyme processes are of high interest for future economies. We present a comprehensive evaluation method for these processes by dividing them into 7 distinct phases, describing them with key performance indicators (KPIs), and summarizing all phases by meta KPIs. Only taking into account the entire process enables identification of weak points and a fair comparison between immobilization methods. We demonstrate the method in two case studies.

Abstract

Enzyme immobilization plays a fundamental role in improving the industrial application of enzyme catalysis, as it greatly influences catalyst stability, reusability, and process control. However, due to the complexity of enzyme production and the variety of different immobilization strategies, research often focuses on isolated parts of the overall process, making an overall comparison of different production and immobilization strategies difficult. This study aims to present a structured, comprehensive method for the evaluation and comparison of immobilized enzyme processes. We identified 7 distinct process phases, each described by key performance indicators (KPIs), and showcase the evaluation in two case studies. In order to gain a complete insight, we then defined and calculated the meta KPIs “recovered activity efficiency”, “space time activity”, “total volumetric turnovers”, and “total process productivity” by assessing the formally calculated KPIs for an efficiency-based evaluation approach. We also utilized the E Factor analysis as a sustainability-based evaluation approach to estimate environmental impacts. We showed that only a holistic view of all phases, by comparison of meta KPIs, allows for accurate comparison of the processes. Additionally, the structured evaluation can be used as a tool for the identification of weak points in each process to elucidate paths for improvement.

Fungal biotechnology plays a vital role in advancing sustainability by offering innovative solutions for resource efficiency, environmental protection, and health improvements. Fungal systems are highly adaptable compared with other biotechnologies, with unique genomic and metabolic functions that enable the large-scale production of valuable compounds. This review emphasizes how fungal biotechnology contributes to global sustainability goals, particularly through artificial intelligence (AI)-driven methods that accelerate strain optimization and metabolic engineering. Engineered Aspergillus strains, with enhanced enzyme production, and Neurospora, a model organism, demonstrate significant potential for industrial applications. These advancements offer cost-effective and resource-efficient solutions, underscoring the importance of interdisciplinary collaboration in fungal biology, genomics, enzymes, and computational approaches to scale fungal biotechnology for sustainable outcomes.

Carbonic anhydrase (CA) enzymes hold strong potential in new biotechnological strategies for accelerated CO2 capture and conversion. Some CAs naturally tolerate the harsh conditions associated with carbon capture technologies; however, long-term durability, while maintaining high activity, presents significant challenges. This review offers an in-depth analysis of the CA enzymes that have been investigated for industrial carbon capture processes and highlights the key amino acids and structural features that are crucial for CA activity and stability under harsh conditions. It examines the impact of site-directed protein engineering to enhance CA efficacy and immobilization strategies. Furthermore, it addresses the challenges of scaling up CA-based technologies and offers strategies to improve their functionality. Future research directions, including artificial intelligence (AI)-driven optimization, are also discussed.

To address the critical bottleneck in the industrial production of sulfonated products, an enhanced synthesis pathway for the common sulfo donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) was developed. Through key enzyme mining and engineering, an efficient microbial platform was established for endogenous PAPS generation and de novo synthesis of sulfonated products.

Nature Synthesis, Published online: 04 July 2025; doi:10.1038/s44160-025-00826-3

Commercial lutein is mainly extracted from marigold flowers, which is an inefficient process. Now a gram-per-litre-scale process for microbial production of lutein is reported using metabolically engineered Corynebacterium glutamicum. By integrating flux optimization, engineered cytochrome P450 enzymes, electron-channelling scaffolds and fermentation strategies, this approach provides a microbial platform for lutein biosynthesis.

Nature Catalysis, Published online: 04 July 2025; doi:10.1038/s41929-025-01350-5

The scope of Lewis acid catalysis mediated by enzymes is low compared with the range of reactions it drives in organic synthesis. Now the substitution of the iron centre with copper, and the subsequent directed evolution, enabled a non-haem iron hydroxylase to efficiently catalyse asymmetric abiotic Conia-ene cyclizations.

Nature Catalysis, Published online: 04 July 2025; doi:10.1038/s41929-025-01372-z

The intermolecular addition of O-centred radicals to alkenes is a challenging endeavour in synthetic chemistry. Now ene-reductases are used to tame reactive O-radicals for intermolecular and enantioselective radical hydroalkoxylation involving a ground-state single-electron radical mechanism.

Flavin's spectroscopic, photophysical, and redox properties are sensitive to its interactions with neighboring polar or charged groups. Flavoproteins capitalize on this sensitivity to tune their chemical reactivity and photochemistry. A fundamental understanding of this tuning mechanism is necessary for the design of novel flavoproteins. Photoactive flavoproteins such as Light-Oxygen-Voltage (LOV) domains have served as important scaffolds to tune photophysics through sequence mutations, resulting in a series of engineered LOV-based proteins that optimize fluorescence, intersystem crossing (ISC), photoreduction, and/or adduct formation over a range of timescales. To better guide future engineering efforts, we have recently employed hybrid quantum mechanical / molecular mechanical (QM/MM) models of LOV domains to study how intradomain electrostatics exert control over flavin's photophysics. In this work, we focus on a series of LOV1 and LOV2 domains from Arabidopsis thaliana (AtLOV), Avena sativa (AsLOV), and Chlamydomonas reinhardtii (CrLOV); by simulating their spectroscopic properties, relative energetics of low-lying singlet and triplet π,π* and n,π* states, and electrostatic projection maps, we present a comparative study that sheds light on the variations in LOV domain's ISC efficiency in these three organisms. We found that for LOV1 , unlike LOV2, the triplet n,π* (TnN,π*) excited state is higher in energy relative to the first optically active singlet π,π* (S1π,π*) state, which corroborates the statement of various literature that ISC is typically less efficient in LOV1 than LOV2 domains. We also find that unfavorable triplet state energetics can explain why CrLOV2 can form the adduct directly from the S1π,π* singlet state.

Nanoparticles (NPs) are known to enhance the activity of enzymes, but such findings remain largely empirical, lacking predictive design principles. Here, we introduce the first high-throughput platform for the discovery of surface-engineered nanoparticles (SENs) that modulate enzyme function. Guided by the hypothesis that surface ligands are primary drivers of activity enhancement, we synthesized a library of 194 gold- and palladium-based SENs functionalized with diverse peptide ligands. These SENs were screened against three model enzymes: cytochrome c, lactoperoxidase (LPO), and lipase. Multiple SENs substantially increased enzymatic activity, with the most effective achieving ~19-fold increase. The resulting dataset enabled the training of a machine learning model that identified key ligand features associated with high-performing SENs, establishing a predictive framework for designing activity-enhancing NPs. Mechanistic studies confirmed that the ligand shell plays a dominant role in driving enhancement, suggesting that effective ligands identified via this approach can be readily transferred across NP platforms. To demonstrate functional relevance, we show that an optimized SEN/LPO pair outperforms LPO in inhibiting the growth of multidrug-resistant bacteria and disrupting biofilm formation. Collectively, this work offers a scalable and generalizable method to map and harness nanoscale structure-function relationships at biointerfaces, with applications in biocatalysis, biosensing, and beyond.

Natural Diels–Alderases help catalyze [4+2] cycloadditions by preorganizing substrates into reactive conformations. However, the role of other catalytic factors, such as electrostatic effects, remain elusive. Here, we combine conceptual Density Functional Theory (CDFT) descriptors and electric field analysis to unravel the electrostatic basis of activity in the Diels-Alderase AbyU. Previously, four different enzyme-substrate poses were identified by docking, of which two showed catalytically favorable free energy barriers based on quantum mechanical/molecular mechanical (QM/MM) reaction simulations. Here, we show that atom-condensed Fukui functions can predict the reactivity based on reactant conformations only, based on the diene carbons involved in bond formation. The importance of the enzyme-diene interaction is supported by electric field analysis, which shows how reactivity of enzyme-substrate poses correlates with aligment of the enzyme field along the diene moiety. Our findings establish a basis for predicting and engineering Diels–Alderase activity based on electrostatic and electronic reactivity features.

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover - i.e., extending lifespan - of short-lived enzymes is thus a rational strategy to conserve energy and carbon, and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain life-shortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4{Delta} strain to raise HDH protein abundance (a proxy for lifespan) by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDHs carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme lifespan in vivo in addition to, as expected, altering kinetic properties.

Metal-Installer enables the rational design of metal-binding sites within pre-existing protein scaffolds. By combining geometric constraints and probability density maps derived from curated metalloprotein datasets, the tool identifies residues suited for mono- and dinuclear coordination with first-row transition metals across the specified ligand sets. Using Metal-Installer, we successfully generated 13 artificial metalloproteins, some of which exhibit the characteristic spectroscopic features of natural metalloenzymes or metal-dependent catalytic activity. These results establish a broadly applicable and accurate strategy for metalloprotein design.

Lavandulol and its ester derivative, lavandulyl acetate, are key fragrance constituents of lavender essential oil with widespread applications in cosmetics, perfumery, and food. However, traditional plant extraction suffers from low yield and unsustainable practices, and chemical synthesis relies on petrochemical feedstocks. Here, we report the first de novo microbial biosynthesis of lavandulol and lavandulyl acetate in Escherichia coli through modular pathway engineering. By screening 13 pyrophosphatases from E. coli, we identified RdgB as an efficient pyrophosphatase catalyzing the conversion of lavandulyl diphosphate into lavandulol, enabling its production at 24.9 mg/L. Building on this, we established a three-plasmid expression system and introduced a lavender-derived alcohol acyltransferase, LiAAT4, to achieve the biosynthesis of lavandulyl acetate at 42.4 mg/L. This study establishes the biosynthetic route to lavandulol in E. coli, demonstrating a viable and sustainable microbial platform for producing significant natural components of lavender oil. Our work provides the basis for future strain optimization and industrial-scale biomanufacturing of high-value monoterpene fragrance molecules.

N-Acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase that can install a {beta}1,4-linked N-acetylglucosamine (GlcNAc) residue at the central {beta}-mannoside of N-glycans. The resulting so-called bisecting GlcNAc is not further extended by glycosyl transferases and has been implicated a wide range of biological processes. The molecular mechanisms by which bisection modulates the biosynthesis of N-glycans and influences molecular recognition is not well understood, which is due to a lack of well-defined N-glycans with and without bisection. We describe a chemoenzymatic methodology that can readily provide a wide range of asymmetrical bisecting bi-, tri- and tetra-antennary N-glycans. It was found GnT-III can act on bi-, tri- and tetra-antennary N-glycans and can also accepts N-glycans having a {beta}1,2GlcNTFA or GlcN3 moiety at the 1,2Man- or 1,6Man-antenna making it possible to prepare panels of asymmetrical N-glycans with and without bisection and having different patterns of sialylation and fucosylation. Kinetic experiments showed GnT-III preferentially modifies bi-antennary glycans. The compounds were printed as a glycan microarray, which was screened for lectin binding. It was found that some lectins preferentially bind to bisecting glycans, whereas others do not tolerate or are not affected by this modification. We investigated receptor specificities of human H1N1 and H2N3 influenza viruses and animal H5N1 viruses that pose a pandemic threat including a virus that has become endemic in cattle. The H1N1 and H2N3 viruses did not tolerate bisection whereas it did not affect H5N1 viruses. A/bovine had the broadest receptor specificity providing a rationale for its wide host range.

The active site architecture of heme enzymes has evolved to control the formation of highly reactive intermediates for oxidative catalysis. Finely tuned proton delivery to heme is essential, yet the mechanisms of proton delivery and the sources of protons are poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase enzyme (ascorbate peroxidase) employing a range of computational molecular modeling approaches, including molecular dynamics (MD) simulations, density functional theory (DFT) and combined quantum mechanics/molecular mechanics (QM/MM) calculations, enhanced sampling techniques, and local electric field (LEF) analyses. Our results show that networks of active site water molecules facilitate proton ex-change with Arg38, which may serve as a transient proton carrier at the γ-edge of the heme, where the substrate (ascorbate) binds. The distal His42 residue aids proton transfer into the active site via solvent from the δ-heme edge. MD simulations of three peroxidases identify hydrated channels in the structure – pointing towards both the γ- and δ-edges of the heme – through which protons from solvent can access the active site. Comparison with structures of 8 other heme peroxidases shows that these channels are conserved features. LEF calculations show a continuous electrostatic funnel, running from positive to negative, that pulls protons from the protein surface along the channels towards the heme iron from both the γ- and δ-heme edges. This electric field is also found to be a conserved feature in several peroxidases; its direction and shape is conserved but the gradient varies between differ-ent enzymes, suggesting that nature preorganises the electric field as a determinant of relative activity. Rather than a simplistic model for proton delivery (in which a single substrate provides a single proton (and an electron) to the heme) the data instead show conserved solvent channels and electrostatic funnels, which provide multiple well defined routes for proton delivery in peroxidase catalysis.

Anaerobic ammonium-oxidizing (anammox) bacteria employ a unique, hydrazine-based pathway to obtain energy from nitrite and ammonium. These organisms express distinct Rieske/ cytochrome b complexes of which the exact function in anammox metabolism is unclear, but which has been proposed to include the generation of NAD(P)H. This would require energetics and structural features unusual for such complexes. Here we present crystal structures and electrochemical investigations of the Rieske subunits of two of these complexes from the anammox organism Kuenenia stuttgartiensis, Kuste4569 and Kustd1480. Both proteins display high redox potentials (>+300 mV), which can be in part explained by their crystal structures and which fit perfectly in the energetic scheme of the proposed NAD(P)H generation mechanism. Moreover, AlphaFold3 models of the parent complexes trace out a path for the electrons required for NAD(P) production, which includes a proposed, novel b-type heme in the membrane-bound part of the complex.