Exploration of three Dyadobacter fermentans enzymes uncovers molecular activity determinants in CE15.

Glucuronoyl esterases (GEs) are serine-type hydrolase enzymes belonging to carbohydrate esterase family 15 (CE15), and they play a central role in the reduction of recalcitrance in plant cell walls by cleaving ester linkages between glucuronoxylan and lignin in lignocellulose. Recent studies have suggested that bacterial CE15 enzymes are more heterogeneous in terms of sequence, structure, and substrate preferences than their fungal counterparts. However, the sequence space of bacterial GEs has still not been fully explored, and further studies on diverse enzymes could provide novel insights into new catalysts of biotechnological interest. To expand our knowledge on this family of enzymes, we investigated three unique CE15 members encoded by Dyadobacter fermentans NS114T, a Gram-negative bacterium found endophytically in maize/corn (Zea mays). The enzymes are dissimilar, sharing ≤ 39% sequence identity to each other' and were considerably different in their activities towards synthetic substrates. Combined analysis of their primary sequences and structural predictions aided in establishing hypotheses regarding specificity determinants within CE15, and these were tested using enzyme variants attempting to shift the activity profiles. Together, the results expand our existing knowledge of CE15, shed light into the molecular determinants defining specificity, and support the recent thesis that diverse GEs encoded by a single microorganism may have evolved to fulfil different physiological functions. KEY POINTS: • D. fermentans encodes three CE15 enzymes with diverse sequences and specificities • The Region 2 inserts in bacterial GEs may directly influence enzyme activity • Rational amino acid substitutions improved the poor activity of the DfCE15A enzyme.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding.

The ability of bovine serum albumin (BSA) to form condensates in crowded environments has been discovered only recently. Effects of this condensed state on the secondary structure of the protein have already been unraveled as some aging aspects, but the pseudo-enzymatic behavior of condensed BSA has never been reported yet. This article investigates the kinetic profile of para-nitrophenol acetate hydrolysis by BSA in its condensed state with poly(ethylene) glycol (PEG) as the crowding agent. Furthermore, the initial BSA concentration was varied between 0.25 and 1 mM which allowed us to modify the size distribution, the volume fraction, and the partition coefficient (varying from 136 to 180). Hence, the amount of BSA originally added was a simple way to modulate the size and density of the condensates. Compared with dilute BSA, the initial velocity (vi) with condensates was dramatically reduced. From the Michaelis-Menten fits, the extracted Michaelis constant Km and the maximum velocity Vmax decreased in control samples without condensates when the BSA concentration increased, which was attributed to BSA self-oligomerization. In samples containing condensates, the observed vi was interpreted as an effect of diluted BSA remaining in the supernatants and from the condensates. In supernatants, the crowding effect of PEG increased the kcat and catalytic efficiency. Last, Vmax was proportional to the volume fraction of the condensates, which could be controlled by varying its initial concentration. Hence, the major significance of this article is the control of the size and volume fraction of albumin condensates, along with their kinetic profile using liquid-liquid phase separation.

Computational analysis of 3D printing: Selecting the better among newly released materials.

Resin-based three-dimensional (3D) printing finds extensive application in the field of dentistry. Although studies of cytotoxicity, mechanical and physical properties have been conducted for newly released 3D printing resins such as Crowntec (Saremco), Temporary Crown Resin (Formlabs) and Crown & Bridge (Nextdent), the resistance of these materials to esterases in saliva has not been demonstrated at the molecular level. Therefore, in this study, the binding affinities and stability of these new 3D printing resins to the catalytic sites of esterases were investigated using molecular docking and molecular mechanics with Poisson-Bolzmann and surface area solvation (MM/PBSA) methods after active pocket screening. Toxicity predictions of the materials were also performed using ProTox-II and Toxtree servers. The materials were analyzed for mutagenicity, cytotoxicity, and carcinogenicity, and LD50 values were predicted from their molecular structures. The results indicated that out of the three novel 3D printing materials, Nexdent exhibited reduced binding affinity to esterases, indicating enhanced resistance to enzymatic degradation and possessing a superior toxicity profile.

Bioorthogonal masked acylating agents for proximity-dependent RNA labelling.

RNA localization is highly regulated, with subcellular organization driving context-dependent cell physiology. Although proximity-based labelling technologies that use highly reactive radicals or carbenes provide a powerful method for unbiased mapping of protein organization within a cell, methods for unbiased RNA mapping are scarce and comparably less robust. Here we develop α-alkoxy thioenol and chloroenol esters that function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a platform for spatial analysis of RNA: bioorthogonal acylating agents for proximity labelling and sequencing (BAP-seq). We demonstrate that, by selectively unmasking the enol probe in a locale of interest, we can map RNA distribution in membrane-bound and membrane-less organelles. The controlled-release acylating agent chemistry and corresponding BAP-seq method expand the scope of proximity labelling technologies and provide a powerful approach to interrogate the cellular organization of RNAs.

Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy.

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/ß-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3 n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3 + peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase.

Acetyl esterases belonging to the carbohydrate esterase family 16 (CE16) is a growing group of enzymes, with exceptional diversity regarding substrate specificity and regioselectivity. However, further insight into the CE16 specificity is required for their efficient biotechnological exploitation. In this work, exo-deacetylase TtCE16B from Thermothelomyces thermophila was heterologously expressed and biochemically characterized. The esterase targets positions O-3 and O-4 of singly and doubly acetylated non-reducing-end xylopyranosyl residues, provided the presence of a free vicinal hydroxyl group at position O-4 and O-3, respectively. Crystal structure of TtCE16B, the first representative among the CE16 enzymes, in apo- and product-bound form, allowed the identification of residues forming the catalytic triad and oxyanion hole, as well as the structural elements related to the enzyme preference for oligomers. The role of TtCE16B in hemicellulose degradation was investigated on acetylated xylan from birchwood and pre-treated beechwood biomass. TtCE16B exhibited complementary activity to commercially available OCE6 acetylxylan esterase. Moreover, it showed synergistic effects with SrXyl43 ß-xylosidase. Overall, supplementation of xylan-targeting enzymatic mixtures with both TtCE16B and OCE6 esterases led to a 3-fold or 4-fold increase in xylose release, when using TmXyn10 and TtXyn30A xylanases respectively.

Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

Structure-directed bioengineering the lid1 of cold-adapted Pseudomonas sp. TB11 esterase to boost catalytic capacity.

Structure-guided bioengineering enzymes has been an efficient strategy to obtain biocatalyst with desirable properties. In this study, the cold-adapted esterase from Pseudomonas sp. (CPE) was optimized through bioinformatic-based structured-guided bioengineering on lid1 region. Substitutions of non-conserved Q55 led to noticeable increase in hydrolysis without sacrificing enzyme thermostability, activating effects of Ca2+ and organic solvents. Compared to the wild type, both of Q55V and Q55N among the constructed variants exhibited about a 2.0-fold and 6.5-fold higher hydrolytic activity toward short-chain and long-chain substrates, respectively. In contrast, lid swapping with the lid of Thermomyces lanuginosus lipase reduced the activity and thermostability of CPE. Catalytic kinetics revealed that substitution of Q55 with Y, V, N and R enhanced the substrate affinity of CPE. Hydrolysis by Q55V remarkedly enriched the characteristic flavor components of single cream. The study sheds light on structure-guided bioengineering of lid tailoring cold-adapted esterases with desired catalytic performance to meet the demand from biotechnological applications.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition.

Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single-molecule level, and it is shown that the dissecting alterations of single-molecule esterase activities can serve as an informative platform for activity-based diagnosis.

Transesterification with CE15 glucuronoyl esterase from Cerrena unicolor reveals substrate preferences.

PURPOSE: Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from Cerrena unicolor (CuGE) can reveal the enzyme's preference for the alcohol-part of the ester-bond. METHODS: This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-O-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme's alcohol preferences. CONCLUSION: CuGE preferentially forms the γ-ester from the methyl ester of 4-O-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme's substrate preferences are primarily dictated by the presence of the 4-O-methylation on the glucuronoyl donor, and secondly on the type of alcohol.

Crystal structure of the GDSL family esterase EstL5 in complex with PMSF reveals a branch channel of the active site pocket.

Esterases/lipases from the GDSL family have potential applications in the hydrolysis and synthesis of important esters of pharmaceutical, food, and biotechnical interests. However, the structural and functional understanding of GDSL enzymes is still limited. Here, we report the crystal structure of the GDSL family esterase EstL5 complexed with PMSF at 2.34 Šresolution. Intriguingly, the PMSF binding site is not located at the active site pocket but is situated in a surface cavity. At the active site, we note that there is a trapped crystallization solvent 1,6-hexanediol, which mimics the bound ester chain, allowing for further definition of the active site pocket of EstL5. The most striking structural feature of EstL5 is the presence of a unique channel, which extends approximately 18.9 Å, with a bottleneck radius of 6.8 Å, connecting the active-site pocket and the surface cavity. Replacement of Ser205 with the bulk aromatic residue Trp or Phe could partially block the channel at one end and perturb its access. Reduced enzymatic activity is found in the EstL5 S205W and EstL5 S205F mutants, suggesting the functional relevance of the channel to enzyme catalysis. Our study provides valuable information regarding the properties of the GDSL-family enzymes for designing more efficient and robust biocatalysts.

cazy_webscraper: local compilation and interrogation of comprehensive CAZyme datasets.

Carbohydrate active enzymes (CAZymes) are pivotal in biological processes including energy metabolism, cell structure maintenance, signalling, and pathogen recognition. Bioinformatic prediction and mining of CAZymes improves our understanding of these activities and enables discovery of candidates of interest for industrial biotechnology, particularly the processing of organic waste for biofuel production. CAZy (www.cazy.org) is a high-quality, manually curated, and authoritative database of CAZymes that is often the starting point for these analyses. Automated querying and integration of CAZy data with other public datasets would constitute a powerful resource for mining and exploring CAZyme diversity. However, CAZy does not itself provide methods to automate queries, or integrate annotation data from other sources (except by following hyperlinks) to support further analysis. To overcome these limitations we developed cazy_webscraper, a command-line tool that retrieves data from CAZy and other online resources to build a local, shareable and reproducible database that augments and extends the authoritative CAZy database. cazy_webscraper's integration of curated CAZyme annotations with their corresponding protein sequences, up-to-date taxonomy assignments, and protein structure data facilitates automated large-scale and targeted bioinformatic CAZyme family analysis and candidate screening. This tool has found widespread uptake in the community, with over 35 000 downloads (from April 2021 to June 2023). We demonstrate the use and application of cazy_webscraper to: (i) augment, update and correct CAZy database accessions; (ii) explore the taxonomic distribution of CAZymes recorded in CAZy, identifying under-represented taxa and unusual CAZy class distributions; and (iii) investigate three CAZymes having potential biotechnological application for degradation of biomass, but lacking a representative structure in the PDB database. We describe in general how cazy_webscraper facilitates functional, structural and evolutionary studies to aid identification of candidate enzymes for further characterization, and specifically note that CAZy provides supporting evidence for recent expansion of the Auxiliary Activities (AA) CAZy family in eukaryotes, consistent with functions potentially specific to eukaryotic lifestyles.

Cell wall and immune modulation by Rv1800 (PPE28) helps M. smegmatis to evade intracellular killing.

Rv1800 is predicted as PPE family protein found in pathogenic mycobacteria only. Under acidic stress, the rv1800 gene was expressed in M. tuberculosis H37Ra. In-silico study showed lipase/esterase activity in C-terminus PE-PPE domain having pentapeptide motif with catalytic Ser-Asp-His residue. Full-length Rv1800 and C-terminus PE-PPE domain proteins showed esterase activity with pNP-C4 at the optimum temperature of 40 °C and pH 8.0. However, the N-terminus PPE domain showed no esterase activity, but involved in thermostability of Rv1800 full-length protein. M. smegmatis expressing rv1800 (MS_Rv1800) showed altered colony morphology and a significant resistance to numerous environmental stresses, antibiotics and higher lipid content. In extracellular and membrane fraction, Rv1800 protein was detected, while C terminus PE-PPE was present in cytoplasm, suggesting the role of N-terminus PPE domain in transportation of protein. MS_Rv1800 infected macrophage showed higher intracellular survival and low production of ROS, NO and expression levels of iNOS and pro-inflammatory cytokines, while induced expression of the anti-inflammatory cytokines. The Rv1800, PPE and PE-PPE showed antibody-mediated immunity in MDR-TB and PTB patients. Overall, these results confirmed the esterase activity in the C-terminus and function of N-terminus in thermostabilization and transportation; predicting the role of Rv1800 in immune/lipid modulation to support intracellular mycobacterium survival.

Microorganisms that produce enzymes active on biodegradable polyesters are ubiquitous.

Biodegradability standards measure ultimate biodegradation of polymers by exposing the material under test to a natural microbial inoculum. Available tests developed by the International Organization for Standardization (ISO) use inoculums sampled from different environments e.g. soil, marine sediments, seawater. Understanding whether each inoculum is to be considered as microbially unique or not can be relevant for the interpretation of tests results. In this review, we address this question by consideration of the following: (i) the chemical nature of biodegradable plastics (virtually all biodegradable plastics are polyesters) (ii) the diffusion of ester bonds in nature both in simple molecules and in polymers (ubiquitous); (iii) the diffusion of decomposers capable of producing enzymes, called esterases, which accelerate the hydrolysis of esters, including polyesters (ubiquitous); (iv) the evidence showing that synthetic polyesters can be depolymerized by esterases (large and growing); (v) the evidence showing that these esterases are ubiquitous (growing and confirmed by bioinformatics studies). By combining the relevant available facts it can be concluded that if a certain polyester shows ultimate biodegradation when exposed to a natural inoculum, it can be considered biodegradable and need not be retested using other inoculums. Obviously, if the polymer does not show ultimate biodegradation it must be considered recalcitrant, until proven otherwise.

Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans.

In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.

A covalent crosslinking strategy to construct a robust peptide-based artificial esterase.

Peptide-based artificial enzymes derived from the supramolecular assembly of short peptides have attracted growing attention in recent years. However, the stability of these artificial enzymes is still a problem since their noncovalent supramolecular structure is quite sensitive and frail under environmental conditions. In this study, we reported a covalent crosslinking strategy for the fabrication of a robust peptide-based artificial esterase. Inspired by the di-tyrosine bonds in many natural structural proteins, multi-tyrosines were designed into a peptide sequence with histidine as the catalytic residue for the ester hydrolysis reaction. Upon the photo-induced oxidation reaction, the short peptide YYHYY rapidly transferred into nanoparticle-shaped aggregates (CL-YYHYY) and displayed improved esterase-like catalytic activity than some previously reported noncovalent-based artificial esterases. Impressively, CL-YYHYY showed outstanding reusability and superior stability under high temperature, strong acid and alkaline and organic solvent conditions. This study provides a promising approach to improving the catalytic activity and stability of peptide-based artificial enzymes.