Biology subjects

Westh, P.

Publications and source records attributed to Westh, P..

11 recordsLinked to original sources

bioRxiv2026

EnZight: A Structure-Guided Algorithm to Identify and Prioritize Substitution Hotspots for Enzyme Engineering

Homologous protein structures contain valuable information about tolerated sequence variation. However, translating this information into practical enzyme design strategies remains challenging. Here we present EnZight, a user-friendly web server that integrates homologous structural alignment with intuitive visualization to identify substitution hotspots in protein cores. EnZight exploits structurally aligned homologs to identify positions where the surrounding structural environment is conserved while the residue at the position varies across homologs. This enables prediction of substitutions that preserve fold integrity while modulating function and thermostability. The approach further provides interactive structural outputs that allow users to inspect and prioritize substitutions manually. To validate the use of EnZight, we used a polyurethane-degrading amidase as a proof of concept. We constructed 34 variants, and 97% were successfully expressed, indicating high foldability of the predicted substitutions. Several substitutions improved both catalytic turnover and thermostability, and, importantly, beneficial substitutions combined additively, enabling stepwise accumulation of improvements. The best triple mutant variant exhibited a six-fold increase in catalytic turnover and 2{degrees}C increase in apparent melting temperature. Enhanced activity toward the pharmaceutical micropollutant flutamide further demonstrates EnZight's broad applicability in identifying substitutions that enable enzyme optimization across diverse substrates.

bioengineering↗

bioRxiv2026

Mapping bacterial cutinase sequence space by high-throughput screening reveals that PET hydrolysis is a rare property

Poly(ethylene terephthalate) (PET) is one of the most widely produced plastics, and enzymatic depolymerization offers a promising route to closed-loop recycling under mild conditions. However, most known bacterial PET hydrolases belong to a conserved canonical-fold cutinase family, leaving much of alpha/beta-hydrolase diversity unexplored. Here, we mapped bacterial cutinase sequence space by combining bioinformatics-guided sequence selection with high-throughput secretion screening in Bacillus subtilis. A library of 1,120 genes encoding 954 unique bacterial cutinases, spanning canonical- and minimal-fold families, was screened for activity on Impranil DLN and semicrystalline PET. We identified 156 secreted cutinases with polyester activity, broadly distributed across sequence space, but only ten showed detectable PET hydrolysis, all from the canonical-fold family. These PET hydrolases were active at 40-50{degrees}C, preferred alkaline pH, and showed moderate thermostability. Our results demonstrate that PET activity is rare among bacterial cutinases and provide a scalable workflow for discovering diverse enzyme starting points.

biochemistry↗

bioRxiv2026

The Thermodynamics of Biomolecular CO2 Capture:Disentangling Equilibria in Amino-Acid-based Systems

Amino acids and peptides are promising building blocks for aqueous biomolecular CO2 capture systems, yet the coupled thermodynamics governing carbamate formation, proton transfer, carbonate speciation, and hydration remain difficult to resolve experimentally. Here, we establish isothermal titration calorimetry (ITC) as a quantitative platform for characterizing these coupled processes by integrating calorimetry with pH titrations, NMR spectroscopy, and a mechanistic thermodynamic model. Using L-lysine, L-arginine, and a series of Lys- and Arg-containing peptides, global fitting of ITC thermograms yielded thermodynamic parameters describing protonation and carbamate formation that accurately reproduced independent pH titrations and NMR-derived speciation. The analysis revealed that the characteristic biphasic calorimetric response originates from the coupled carbonate-amine equilibrium network and buffer collapse rather than carbamate saturation. Lys formed -, {varepsilon}-, and ,{varepsilon}-dicarbamates and exhibited more favorable apparent carbamate thermodynamics than Arg with the {varepsilon}-carbamate lying among the most favorable carbamate-forming amine sites reported for aqueous amines. Model-guided exploration of the fitted thermodynamic landscape further demonstrated that maximizing total CO2 retention, amine-mediated capture, and carbamate formation are distinct optimization problems governed by different combinations of pH, temperature, and CO2 loading. Extension to systematically spaced Lys-containing peptides showed that inter-amine separation alone does not control carbamate stability, highlighting the dominant role of the local thermodynamic environment in biomolecular CO2 capture. This work establishes ITC as a powerful experimental approach for extracting CO2-amine thermodynamics and provides a predictive framework for the rational design and optimization of amino acid-, peptide-, and protein-based carbon capture systems.

biochemistry↗

bioRxiv2026

Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of five novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Two candidate enzymes, u17 u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Among them, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. To further assess enzymatic performance on a realistic industrial material, the panel was then tested on a generic flexible foam substrate derived from 2,4- and 2,6-toluene diisocyanate (TDA), where u15 and u17 emerged as the most active candidates. Collectively, we benchmark the structural framework presented by enzymes in the amidase signature family as a strong foundation for further optimization aiming at advancing sustainable and scalable biocatalytic recycling of polyurethanes.

biochemistry↗

bioRxiv2025

A high throughput investigation of the binding specificity of carbohydrate-binding modules for synthetic and natural polymers

Carbohydrate-binding modules (CBMs) are non-catalytic domains that enhance enzyme binding to substrates, crucial for polysaccharide degradation. Type A CBMs also show potential for engineering plastic-degrading enzymes due to their high affinity for synthetic polymers. This study presents a high-throughput screening pipeline for characterizing the affinity and specificity of Type A CBMs to the synthetic polymers polyethylene terephthalate (PET), polystyrene (PS), and polyethylene (PE), and the natural polysaccharides cellulose, chitin, and starch. Approximately 800 CBMs from the families CBM2, CBM3, CBM10, and CBM64 were expressed as enhanced green fluorescent protein (EGFP)-fusion proteins and tested for binding using a modified holdup assay, which could produce up to 10,000 data points per day. The screening identified approximately 150 binders for PET and PE, around 250 for PS, and demonstrated family-specific binding patterns for avicel, chitin, and starch. Distinct patterns of substrate affinity and specificity were observed, which allowed for rationalizations of binding at the structural and phylogenetic levels. To demonstrate practical utility, CBMs with high PET affinity were fused to the PET hydrolase LCCICCG, enhancing enzymatic activity on crystalline PET powder by around 5-fold. Importantly, these CBM-enzyme fusions mitigated competitive binding to inert plastic impurities, improving performance in mixed plastic assays. This work significantly expands the known repertoire of CBMs capable of binding synthetic polymers, advances our understanding of CBM-substrate interactions, and provides knowledge for engineering enzymes to tackle plastic pollution, particularly in municipal solid waste where mixed plastics pose significant challenges. Significance StatementImproper recycling of plastic poses a severe environmental threat, with synthetic polymers persisting in ecosystems for decades. Enzymatic recycling of plastic offers a promising solution, with optimization of the enzymes and processes ongoing. To expand the toolkit for engineering enzymes via substrate-binding module fusion, we developed a high-throughput screening platform to identify carbohydrate-binding modules (CBMs) that selectively bind synthetic plastics such as PET, polystyrene, and polyethylene, as well as natural polysaccharides. By screening 797 CBMs, we discovered binding domains with distinct specificities, enabling the design of enzyme fusions that enhance plastic degradation, even in mixed plastic environments. This approach provides insights into protein-substrate interactions that can be leveraged for both waste management and bioengineering innovations.

bioengineering↗

bioRxiv2024

Interrelationship of substrate crystallinity, enzyme binding strength, and cellulase activity

Structural polysaccharides are difficult to degrade due to their crystalline structure. Hence, industrial conversion of biomass has focused on both substrate pretreatment and enzyme engineering to improve the biochemical conversion of biomass into fuels and chemicals. However, few studies have explored the interrelationship between substrate crystallinity and cellulase activity. Here, we systematically investigated the kinetics of structurally diverse cellulases on five cellulosic substrates with varying crystallinity. Regardless of enzyme structure and catalytic mechanism, we observed a linear scaling of the kinetic parameters (KM and kcat) in a log-log plot, indicating a linear free energy relationship (LFER) between binding and activation energy. LFERs were observed for all investigated substrates, but their slopes varied distinctly and appeared linked to the substrate crystallinity. Substrates with low crystallinity exhibited LFERs with a slope near 1, while highly crystalline substrates had a slope of approximately 0.25, providing insights into the transition state (TS) for the rate-limiting step. We propose that maximal turnover was limited by slow dissociation, with the TS structurally close to the enzyme-ligand complex on crystalline substrate, while on amorphous substrate, the TS structure was closer to the dissociated system. We suggest that these observations reflect competing interactions of the ligand with respectively the enzyme binding cleft and the substrate matrix. This study emphasizes the interconnected nature of substrate pretreatment and enzyme engineering, urging a holistic approach to propel the biochemical conversion of lignocellulosic biomass, crucial for advancing sustainable production of fuels and chemicals.

biochemistry↗

bioRxiv2023

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as kinetic parameters for soluble PET fragments. The results indicate that a proficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product occurs here. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

biochemistry↗

bioRxiv2022

Reaction pathways for the enzymatic degradation of poly(ethylene terephthalate): What characterizes an efficient PET-hydrolase?

Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model, and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (kcat/KM-values of about 105 M-1s-1) in the aqueous bulk. This meant that about 70% of the final, monoaromatic products was formed via soluble di-or tri-aromatic intermediates.

biochemistry↗

bioRxiv2022

Sabatier principle for rationalizing enzymatic hydrolysis of a synthetic polyester

Interfacial enzyme reactions are common in nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure-function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium T. fusca (TfC) when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme-PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength is in accordance with the Sabatier principle. One consequence of these results is that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of kcat at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum and the turnover rate was 5-fold improved at weakened substrate binding. Our results show that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding, since strong adsorption seems to limit their catalytic performance. ToC graphics O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Comparative biochemistry of four polyester (PET) hydrolases

The potential of bioprocessing in a circular plastic economy has strongly stimulated research in enzymatic degradation of different synthetic resins. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic degrading enzymes) acting on the insoluble substrate, has not been established. Here, we propose such a framework and test it against kinetic measurements on four PET hydrolases. The analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional units of M-1s-1. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. We found that the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions, and that this relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, while the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET, when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.

biochemistry↗

bioRxiv2020

Physical constrains and functional plasticity of cellulases: Linear scaling relationships for a heterogeneous enzyme reaction

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.

biochemistry↗