Polyester degradation by soil bacteria: identification of conserved BHETase enzymes in Streptomyces.

The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.

Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

BACKGROUND: 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. METHODS AND RESULTS: 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 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-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 20 min 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/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: 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.

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.

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate.

The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0-9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and kcat/Km (598.57 ± 19.97 s-1mM-1) for pN-C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The Km, kcat, and kcat/Km values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s-1, and 2.67 ± 0.02 mM-1 s-1, respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s-1, and 0.02 ± 0.001 mM-1 s-1, respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.

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.

Discovery and mechanism explanation of a novel green biocatalyst esterase Bur01 from Burkholderia ambifaria for ester synthesis under aqueous phase.

Biocatalyst catalyzing the synthesis of esters under aqueous phase is an alternative with green and sustainable characteristics. Here, a biocatalyst esterase Bur01 was identified through genome sequencing and gene library construction from a Burkholderia ambifaria BJQ0010 with efficient ester synthesis property under aqueous phase for the first time. Bur01 was soluble expressed and the purified enzyme showed the highest activity at pH 4.0 and 40 °C. It had a broad substrate spectrum, especially for ethyl esters. The structure of Bur01 was categorized as a member of α/ß fold hydrolase superfamily. The easier opening of lid under aqueous phase and the hydrophobicity of substrate channel contribute to easier access to the active center for substrate. Molecular docking and site-directed mutation demonstrated that the oxyanion hole Ala22, Met112 and π-bond stacking between His24 and Phe217 played essential roles in catalytic function. The mutants V149A, V149I, L159I and F137I enhanced enzyme activity to 1.42, 1.14, 1.32 and 2.19 folds due to reduced spatial resistance and increased hydrophobicity of channel and ethyl octanoate with the highest conversion ratio of 68.28 % was obtained for F137I. These results provided new ideas for developing green catalysts and catalytic basis of mechanistic studies for ester synthetase under aqueous phase.

Determining the esterase activity of peptides and peptide assemblies.

Catalytic peptides are gaining attention as alternatives to enzymes, especially in industrial applications. Recent advances in peptide design have improved their catalytic efficiency with approaches such as self-assembly and metal ion complexation. However, the fundamental principles governing peptide catalysis at the sequence level are still being explored. Ester hydrolysis, a well-studied reaction, serves as a widely employed method to evaluate the catalytic potential of peptides. The standard colorimetric reaction involving para-nitrophenyl acetate hydrolysis acts as a benchmark assay, providing a straightforward and efficient screening method for rapidly identifying potential catalysts. However, maintaining standardized conditions is crucial for reproducible results, given that factors such as pH, temperature, and substrate concentration can introduce unwanted variability. This necessity becomes particularly pronounced when working with peptides, which often exhibit slower reaction rates compared to enzymes, making even minor variations significantly influential on the final outcome. In this context, we present a refined protocol for assessing the catalytic activity of peptides and peptide assemblies, addressing critical considerations for reproducibility and accuracy.

Cascade Mesophase Transitions of Multi-enzyme Responsive Polymeric Formulations.

Studying how synthetic polymer assemblies respond to sequential enzymatic stimuli can uncover intricate interactions in biological systems. Using amidase- and esterase-responsive PEG-based diblock (DBA) and triblock amphiphiles (TBAs), we created two distinct formulations: amidase-responsive DBA with esterase-responsive TBA and vice versa. We studied their cascade responses to the two enzymes and the sequence of their introduction. These formulations underwent cascade mesophase transitions upon the addition of the DBA-degrading enzyme, transitioning from (i) coassembled micelles to (ii) triblock-based hydrogel, and ultimately to (iii) dissolved polymers when exposed to the TBA hydrolyzing enzyme. The specific pathway of the two mesophase transitions depended on the compositions of the formulations and the enzyme introduction sequence. The results highlight the potential for designing polymeric formulations with programmable multistep enzymatic responses, mimicking the complex behavior of biological macromolecules.

A Novel Thermo-Alkaline Stable GDSL/SGNH Esterase with Broad Substrate Specificity from a Deep-Sea Pseudomonas sp.

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000 m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4 kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60 °C, respectively, but it retained more than 50% of its activity over the pH range of 7-11 and temperature range of 10-75 °C. In addition, EstGoM was tolerant of up to 1 M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and ß-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications.

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.

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.

Efficient activity screening of new glucuronoyl esterases using a pNP-based assay.

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-ß-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (ß-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-ß-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.

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.

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.

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.

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.