Efficient and durable catalysts are essential for enhancing the rate of CO2 hydration in aqueous capture systems and addressing the environmental challenges posed by rising atmospheric CO2 levels. Traditional capture methods that rely on strongly alkaline solvents face substantial drawbacks, including high regeneration energy costs and safety concerns owing to harmful byproducts. Carbonic anhydrase (CA), a zinc-containing metalloenzyme, catalyzes CO2 hydration at near-diffusion-limited rates but rapidly degrades in real-world environments. Although various CA-mimetic catalysts have been investigated, they frequently exhibit poor durability and structural stability. In this study, we introduce ZnNC900, a robust Zn single-atom nanozyme derived from a zeolitic imidazolate framework (ZIF-8) through controlled carbonization. ZnNC900 exhibited remarkable catalytic activity for the p-nitrophenyl acetate esterase reaction, which was attributed to its CA-resembling single-atomic Zn sites. Furthermore, surface functionalization with amine-terminalized polyethylene glycol improves the colloidal stability and catalytic performance of ZnNC900 by enhancing its hydrophilicity and CO2 affinity. These enhancements highlight the potential of ZnNC900 as a durable and efficient catalyst for aqueous CO2 capture.

Dual-function hemoglobin-encapsulating ZIF-8 nanoparticles: Oxygen transport capability and carbonic anhydrase-like activity

The carbamate pesticide carbaryl poses significant ecological and health risks due to its widespread misuse and improper long-term disposal. In this study, we engineered a novel bacterial carbaryl-degrading esterase to enhance enzymatic activity, thermostability, and carbaryl degradation through semi-rational design and whole-cell biocatalysis strategies. An esterase, designated Est03320, was identified from Bacillus velezensis MB01B. Phylogenetic analysis revealed it resides in a distinct clade. However, it contains a catalytic domain similar to those of the well-characterized carbaryl-hydrolyzing esterase CarH and the chlorpyrifos-degrading esterase MPH. Est03320 was expressed and purified from Escherichia coli BL21(DE3) cells, demonstrating high hydrolytic activity against short-chain p-nitrophenyl esters and carbaryl. The enzyme exhibits an optimum pH of 6.5 and an optimal temperature of 25°C, with a Km of 139.70 µM and a Vmax of 29.47 µM/min. The mutant Est03320-L230G, engineered through a combined semi-rational design and site-directed mutagenesis approach, exhibited a 68.7% increase in enzymatic activity toward the substrate p-nitrophenyl acetate, an extended half-life at 25°C from 13.6h to 40.6h, and a 96.5% enhancement in carbaryl degradation efficiency. To construct a whole-cell biocatalyst, this mutant esterase was further fused with the autotransporter EhaA and displayed on the surface of engineered Pseudomonas sp. MB04R-14-03320-L230G-EhaA cells. The resulting biocatalyst showed high efficiency in degrading and mineralizing carbaryl, retaining over 83% of its activity after five consecutive degradation cycles. This integrated approach significantly enhances both enzymatic performance and reusability, offering a sustainable and effective approach for mitigating carbaryl pollution and advancing environmental protection.

Usefulness of plasma and apolipoprotein B-depleted serum samples in paraoxonase 1 assessment☆

Despite being structurally rudimentary, single amino acids and their derivatives demonstrate a remarkable ability to self-assemble into ordered nanostructures that renders potential catalytic activity. While many reports of catalysis utilizing amyloid-inspired peptide nanostructures are available, single amino acid derived hydrogel-based catalysts are rare. Herein, we report an elementary amino acid derivative, fluorenylmethoxycarbonyl-L-tryptophan (FT), based hydrogel that catalyzed the hydrolysis of p-nitrophenyl acetate, courtesy of the suitable positioning of indole residues in its ordered nanostructures. Exhibiting pathway complexity, the hydrogel nanofibers, initially formed as a kinetically trapped phase, underwent a morphological transition into thermodynamically stable semicrystalline microstructures, displaying better catalytic prowess than the hydrogel. Composite hydrogels with carbon nanomaterials improved the FT gels' mechanical properties and catalytic efficiency, ultimately bestowing enzyme-like hydrolytic activity. The FT hydrogel was finally utilized to engineer nanohybrid gels with gold nanoparticles as an efficient catalyst for dye degradation. The handling scope of these nanohybrid gels, along with morphological control, was improved by preparing core-shell hydrogel beads with alginate, realizing their practical catalytic potential for water remediation. Strikingly, a single hydrogel bead was capable of driving almost complete degradation for both cationic and anionic dyes. This reflects the optimized microenvironment for electron transfer and substrate diffusion within the porous structure of the developed beads. The efficacious catalysis by the bulk hydrogels/beads demonstrates the role of confinement, high surface area, and substrate diffusion through their porous architecture. Moreover, the hydrogels' semisolid nature offered excellent reusability, underscoring their role in sustainable and cost-effective catalytic applications.

Acetic acid production from corn straw via enzymatic degradation using putative acetyl esterase from the metagenome assembled genome.

Due to the inherent limitations of natural enzymes, biomimetic enzymes have received tremendous attention, among which those arising from peptide self-assembly are of particular interest due to their resemblance to natural enzymes in composition and hierarchical structures, as well as their structural robustness and designability. Despite considerable advances achieved in this area, it remains a major challenge to construct active site clefts through peptide self-assembly. Here, we report the design of polar zippers between peptide β-sheets to mimic the catalytic microenvironment of natural enzymes. As a supersecondary structural motif stabilized by the side chain-side chain hydrogen bonding, polar zippers not only promote significant β-sheet lamination to form wide nanoribbons but also constitute clefts on the nanoribbons' surface. Among the three designed peptide analogues (I3GH, I3GHK, and I3HGK), histidine (His or H) polar zippers between β-sheets form only in the self-assembly of I3HGK, thus leading to the formation of wide I3HGK nanoribbons and thin I3GH and I3GHK nanofibrils. Compared to the I3GHK and I3GH nanofibrils, the I3HGK nanoribbons exhibit substantially increased catalytic efficiency in the hydrolysis of p-nitrophenyl acetate (pNPA) due to the synergistic interplay of polar reactive His residues and hydrophobic Ile(I) residues buried within the clefts. By substituting other uncharged polar residues for His within the clefts, the catalytic ability of the peptide nanoribbons can be tuned, with the I3CGK ones exhibiting the highest catalytic efficiency in the pNPA hydrolysis, owing to the potent nucleophilicity of the cysteine (Cys or C) side chain. This work offers a new conceptual framework for mimicking the catalytic cleft of natural enzymes through the rational design and self-assembly of short peptides.

Peptide-based nanomolecular constructs offer great possibilities for designing catalytic molecular systems mimicking enzymes. In this study, we designed three tripeptide catalysts that can possibly mimic hydrolase enzymes, with the objective of systematically verifying the scope of modulating enzymatic activity. Histidine residue was placed at three different locations in Fmoc-tripeptide sequences, thus generating three chemically similar but sequentially different molecules, P1, P2, and P3. From our study, the peptide catalyst P3 has shown maximum catalytic activity with a chromogenic substrate, p-nitrophenyl acetate, that gets hydrolyzed to p-nitrophenol. The catalytic activity has increased with an increase in pH and temperature, though pH dependency cannot be generalized and can vary depending on the reaction mechanism. Importantly, this study successfully demonstrates the possibility of modulating the activity of functional mimics of bioactive molecules by tuning the principal components of functional molecules.

Design strategies for novel esterase purification processes from Trichoderma harzianum-An insight into kinetic and thermodynamic analyses.

Probing the effect of glycation on the pseudo-esterase activity of Human Serum Albumin.

Enzymatic biodegradation of Poly(ε-Caprolactone) (PCL) by a thermostable cutinase from a mesophilic bacteria Mycobacterium marinum.

Abstract In Indonesia, about 83% of plastic waste is mismanaged, causing significant harm to ecosystems. Carboxylic ester hydrolases (CEHs)-producing bacteria offer a sustainable solution by degrading plastics through ester bond hydrolysis. CEHs are chosen for their effective hydrolytic properties and ease of detection. The study aims to investigate the CEHs production activity from several isolated bacteria from landfills to better understand their function in plastic degradation. We hypothesize that these bacteria are capable of producing CEH enzymes. Twelve bacterial isolates were isolated from Bantar Gebang and Cipayung landfills to identify novel species with the highest specific CEHs’ activity. This research identified bacteria with the highest specific enzyme activity by screening them on agar media with different substrates (Tween-20, Tween-80, and Olive oil). A specific activity assay was employed using a model substrate, p-nitrophenyl acetate, because this substrate contains ester bonds that CEHs can break down, mimicking the process of ester bond breakage in some plastics. Bacteria exhibiting the highest activity were revealed through 16S rRNA sequencing. The supernatant from isolates obtained from Cipayung landfill soil, which exhibited the highest specific activities of CEHs at 0.85 ± 0.07 U/mg and 0.81 ± 0.12 U/mg, strongly correlated with the results of agar screening. Through 16S rRNA analysis, these isolates were identified as Pseudomonas aeruginosa strain M4 and Bacillus spp., both of which appeared to produce esterase and lipase enzymes. This research benefits to explore plastic-degrading bacteria isolated from Indonesian landfills and provide a promising solution to the plastic waste problem.

Hierarchically ordered truncated single crystal mesoporous ZIF-8 as a solid platform for lipase immobilization

Data are accumulating on the hydrolytic activity of serum albumin towards esters and organophosphates. Previously, with the help of the technology of proton nuclear magnetic resonance (1H NMR) spectroscopy, we observed the yield of acetate in the solution of bovine serum albumin and p-nitrophenyl acetate (NPA). Thus, we showed that albumin possesses true esterase activity towards NPA. Then, using the methods of molecular docking and molecular dynamics, we established site Sudlow I as the catalytic center of true esterase activity of albumin. In the present work, to expand our understanding of the molecular mechanisms of albumin pseudoesterase and true esterase activity, we investigated-in experiments in vitro and in silico-the interaction of anticoagulant warfarin (WRF, specific ligand of site Sudlow I) and benzodiazepine diazepam (DIA, specific ligand of site Sudlow II) with albumins of different species, and determined how the binding of WRF and DIA affects the hydrolysis of NPA by albumin. It was found that the characteristics of the binding modes of WRF in site Sudlow I and DIA in site Sudlow II of human (HSA), bovine (BSA), and rat (RSA) albumins have species differences, which are more pronounced for site Sudlow I compared to site Sudlow II, and less pronounced between HSA and RSA compared to BSA. WRF competitively inhibits true esterase activity of site Sudlow I towards NPA and does not affect the functioning of site Sudlow II. Diazepam can slow down true esterase activity of site Sudlow I in noncompetitive manner. It was concluded that site Sudlow I is more receptive to allosteric modulation compared to site Sudlow II.

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters

Paraoxonase-1 (PON-1) has been suggested as a marker of inflammation and oxidative stress in horses and could potentially be used for prognostication in horses with colitis. Assessment of PON-1 in horses with colitis and comparison of two methods. Serum PON-1 was measured by two methods (paraoxon and p-nitrophenyl acetate) in 161 horses with colitis and 57 controls. Follow-up samples obtained during hospitalization were available from 106 horses with colitis. The two methods were compared. Serum PON-1 was significantly lower in horses with colitis than in healthy horses (P < .0001 for both methods) as well as in nonsurvivors compared with survivors (P = .0141 [paraoxon-based method] and P < .0001 [p-nitrophenyl acetate-based method]), but with marked overlap between groups. PON-1 activity did not change parallel to a change in inflammatory status in response to treatment when assessed at admission and in up to seven follow-up samples. Admission PON-1 activity could not reliably classify horses as survivors or nonsurvivors, with sensitivity and specificity ranging between 53.1% and 72.9%. Results from the two methods were comparable. Both methods reliably measured serum PON-1 activity. Significant differences in PON-1 activity were found between healthy horses and horses with colitis and between survivors and nonsurvivors. However, PON-1 activity varied considerably within groups. Both the proposed reference intervals as well as alternative cutoff values resulted in suboptimal diagnostic and prognostic performance, and the use of serum PON-1 in horses with colitis thus seems to add little to existing diagnostic and prognostic markers.

House flies Musca domestica L. (Diptera: Muscidae) serve as a common model organism for testing of insecticides and research of insecticidal resistance mechanisms in insects. One of important stages is to assess of detoxifying enzyme activities including carboxylesterase activities (CarE). In this study, we compared specific activities and kinetic parameters (Vmax and Km) of CarE in adults M. domestica of two laboratory strains (TY, UF) depending on the enzymatic substrate used. The specific CarE activities towards α- and β-naphthyl acetate (α-NA and β-NA) were similar in both males and females of the TY strain. In males of the UF strain, the value of the specific and the maximal velocity (Vmax) of β-NA hydrolysis was 1.90- and 1.57-fold respectively less than that of α-NA; this difference was not observed in females of the same strain. Some characteristics of CarE varied depending on sex of insects when p-nitrophenyl acetate was used as an enzymatic substrate. In particular, the specific activity was 1.62-fold less in males of the UF strain compared to this value in females. The activity and main kinetic parameters of CarE towards α-NA not differed statistically significant depending on sex and the strains. Based on the results obtained we suggest that α-naphthyl acetate is the preferred substrate to evaluate the CarE enzymatic activity in the model insect M. domestica of different strains.

Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58s-1mM-1, 1.09s-1mM-1, and 0.062s-1mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20min at 70°C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 μM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 μM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Protein design is central to nearly all protein engineering problems, as it can enable the creation of proteins with new biological functions, such as improving the catalytic efficiency of enzymes. One key facet of protein design, fixed-backbone protein sequence design, seeks to design new sequences that will conform to a prescribed protein backbone structure. Nonetheless, existing sequence design methods present limitations, such as low sequence diversity and shortcomings in experimental validation of the designed functional proteins. These inadequacies obstruct the goal of functional protein design. To improve these limitations, we initially developed the Graphormer-based Protein Design (GPD) model. This model utilizes the Transformer on a graph-based representation of three-dimensional protein structures and incorporates Gaussian noise and a sequence random masks to node features, thereby enhancing sequence recovery and diversity. The performance of the GPD model was significantly better than that of the state-of-the-art ProteinMPNN model on multiple independent tests, especially for sequence diversity. We employed GPD to design CalB hydrolase and generated nine artificially designed CalB proteins. The results show a 1.7-fold increase in catalytic activity compared to that of the wild-type CalB and strong substrate selectivity on p-nitrophenyl acetate with different carbon chain lengths (C2-C16). Thus, the GPD method could be used for the de novo design of industrial enzymes and protein drugs. The code was released at https://github.com/decodermu/GPD.