Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea).

Biosynthesis, stabilization, and storage of carotenoids are vital processes in plants that collectively contribute to the vibrant colors observed in flowers and fruits. Despite its importance, the carotenoid storage pathway remains poorly understood and lacks thorough characterization. We identified two homologous genes, BjA02.PC1 and BjB04.PC2, belonging to the esterase/lipase/thioesterase (ELT) family of acyltransferases. We showed that BjPCs in association with fibrillin gene BjFBN1b control the stable storage of carotenoids in yellow flowers of Brassica juncea. Through genetic, high-resolution mass spectrometry and transmission electron microscopy analyses, we demonstrated that both BjA02.PC1 and BjB04.PC2 can promote the accumulation of esterified xanthophylls, facilitating the formation of carotenoid-enriched plastoglobules (PGs) and ultimately producing yellow pigments in flowers. The elimination of BjPCs led to the redirection of metabolic flux from xanthophyll ester biosynthesis to lipid biosynthesis, resulting in white flowers for B. juncea. Moreover, we genetically verified the function of two fibrillin genes, BjA01.FBN1b and BjB05.FBN1b, in mediating PG formation and demonstrated that xanthophyll esters must be deposited in PGs for stable storage. These findings identified a previously unknown carotenoid storage pathway that is regulated by BjPCs and BjFBN1b, while offering unique opportunities for improving the stability, deposition, and bioavailability of carotenoids.

Quantum Mechanics/Molecular Mechanics Studies on the Catalytic Mechanism of a Novel Esterase (FmtA) of Staphylococcus aureus.

FmtA is a novel esterase that shares the penicillin-binding protein (PBP) core structural folding but found to hydrolyze the removal of d-Ala from teichoic acids. Molecular docking, dynamics, and MM-GBSA of FmtA and its variants S127A, K130A, Y211A, D213A, and K130AY211A, in the presence or absence of wall teichoic acid (WTA), suggest that active site residues S127, K130, Y211, D213, N343, and G344 play a role in substrate binding. Quantum mechanics (QM)/molecular mechanics (MM) calculations reveal that during WTA catalysis, K130 deprotonates S127, and the nucleophilic S127 attacks the carbonyl carbon of d-Ala bound to WTA. The tetrahedral intermediate (TI) complex is stabilized by hydrogen bonding to the oxyanion holes. The TI complex displays a high energy gap and collapses to an energetically favorable acyl-enzyme complex.

Zymography for Picogram Detection of Lipase and Esterase Activities.

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata.

OBJECTIVE: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. MATERIALS AND METHODS: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. RESULTS: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and ß-eterase levels. CONCLUSIONS: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

OBJETIVO: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. MATERIAL Y MÉTODOS: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. RESULTADOS: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y ß-eterasas. CONCLUSIONES: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Forming a Large-Scale Droplet Array in a Microcage Array Chip for High-Throughput Screening.

Forming a large-scale droplet array plays an important role for microfluidic droplet-based high-throughput screening and analysis. Herein, we describe a simple and rapid method to form a large-scale two-dimension (2D) droplet array by using a microcage array chip. Differing from the previous droplet array formation methods, microcages formed by being surrounded by multiple micropillars could rapidly spread the oil phase through the gaps between the micropillars and trap droplets with fast speed and convenient operation. We formed a large-scale 2D monolayer droplet array containing approximately 1 000 000 droplets on a 5.5 cm × 5.5 cm microcage array chip within 90 s. The droplets in the droplet array could be further incubated for performing biochemical reactions and detected by a fluorescence microscope in real time. Due to the exact trapping and positioning functions of the microcages to the droplets, single targeted fluorescent droplets in the array could be individually picked out and transferred to culture medium by a microfluidic droplet-handling robot with a success rate of 100% and a picking operation time of 2.0 s for one droplet under the optimized conditions. This system was validated in the screening of the bacterium expressing the esterase AFEST from a mixture of AFEST-expressing and phosphotriesterase-expressing E. coli cells, achieving a success rate of 100% for single-droplet picking while maintaining the bacterial cell viability. The present system has the potential to be applied in high-throughput screening and analysis, such as single cell analysis, directed evolution, and drug screening.

Metal-Organocatalyst for Detoxification of Phosphorothioate Pesticides: Demonstration of Acetylcholine Esterase Activity.

In recent years, transition metal complexes have been developed for catalytical degradation of a phosphate ester bond, particularly in RNA and DNA; however, less consideration has been given for development of complexes for the degradation of a phosphorothioate bond, as they are the foremost used pesticides in the environment and are toxic to human beings. In this context, we have developed copper complexes of benzimidazolium based ligands for catalytical degradation of a series of organophosphates (parathion, paraoxon, methyl-parathion) at ambient conditions. The copper complexes (assigned as N1-N3) were characterized using single X-ray crystallography which revealed that all three complexes are mononuclear and distorted square planner in geometry. Further, the solution state studies of the prepared complexes were carried out using UV-visible absorption, fluorescence spectroscopy, and cyclic voltametry. The complexes N1 and N2 have benzimidazolium ionic liquid as base attached with two 2-mercapto-benzimidazole pods, whereas complex N3 contains a nonionic ligand. The synthesized copper complexes were evaluated for their catalytic activity for degradation of organophosphates. It is interesting that the complex containing the ionic ligand efficiently degrades phosphorothioate pesticides, whereas complex N3 was not found to be appropriate for degradation due to a weaker conversion rate. The organophosphate degradation studies were monitored by recording absorbance spectra of parathion in the presence of catalyst, i.e., copper complexes with respect to time. The parathion was hydrolyzed into para-nitrophenol and diethyl thiophosphate. Moreover, to analyze the inhibition activity of the pesticides toward acetylcholine esterase enzyme in the presence of prepared metal complexes, Ellman's assay was performed and revealed that, within 20 min, the inhibition of acetylcholine esterase enzyme decreases by up to 13%.

Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit.

We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.

Evaluation of 3 esterase tests for the diagnosis of subclinical mastitis at dry-off and freshening in dairy cattle.

Subclinical mastitis (SCM) and intramammary infection (IMI) increase esterase activity in the glandular secretions of dairy cattle. Our objective was to evaluate the clinical performance of 3 commercially available esterase tests for diagnosing SCM and IMI. Foremilk samples were collected from 380 quarters (96 cows) at dry-off and from 329 quarters (83 cows) within 4 to 7 d after calving. Quarter somatic cell count (SCC) was measured using the reference method (DeLaval cell counter; De Laval International AB, Tumba, Sweden) with SCM defined as SCC >200,000 cells/mL. Bacterial culture of foremilk samples was used to diagnose IMI based on the growth of ≥100 cfu/mL. The SCC was estimated using 3 PortaSCC tests (PortaCheck, Moorestown, NJ) from the measured esterase activity and the California Mastitis Test (CMT). Clinical performance was evaluated using logistic regression to determine the area under the receiver operating characteristic curve (AUC) and identify test sensitivity (Se) and specificity (Sp) at the optimal cut-point for diagnosing SCM and IMI. Test agreement was also evaluated using the kappa coefficient (κ) and weighted κ. The PortaSCC color test was the best-performing PortaSCC test for diagnosing SCM at dry-off (AUC = 0.90, Se = 0.91, Sp = 0.81, κ = 0.71) and at freshening (AUC = 0.86, Se = 0.74, Sp = 0.95, κ = 0.72), at an optimal cut-point of ≥250,000 cells/mL but required 45 min to produce a result. For comparison, the CMT required 2 min to produce a result and a CMT score of trace or higher was superior to the PortaSCC color test for diagnosing SCM at dry-off (AUC = 0.95, Se = 0.95, Sp = 0.86, κ = 0.81) and freshening (AUC = 0.88, Se = 0.79, Sp = 0.95, κ = 0.76). The PortaSCC quick test was the best-performing PortaSCC test for diagnosing IMI at dry-off (AUC = 0.81, Se = 0.81, Sp = 0.78 κ = 0.40) and required 5 min to produce a result, whereas the PortaSCC color test was the best performing PortaSCC test for diagnosing IMI at freshening (AUC = 0.80, Se = 0.75, Sp = 0.79 κ = 0.38). For comparison, the CMT was inferior to the PortaSCC quick test for diagnosing IMI at dry-off (AUC = 0.73, Se = 0.76, Sp = 0.60, κ = 0.20) but was equivalent to the PortaSCC color test at freshening (AUC = 0.79, Se = 0.58, Sp = 0.93, κ = 0.50). The PortaSCC color and quick tests and CMT were considered good tests for diagnosing SCM and IMI because clinically useful tests typically have an AUC >0.80 and κ >0.6. Based on the test sensitivity, cost, and analysis time, there does not appear to be a persuasive reason to select the PortaSCC tests over the traditional CMT for diagnosing SCM and IMI.

Evaluation of droplet-based microfluidic platforms as a convenient tool for lipases and esterases assays.

The accurate estimation of kinetic parameters is of fundamental importance for biochemical studies for research and industry. In this paper, we demonstrate the application of a modular microfluidic system for execution of enzyme assays that allow determining the kinetic parameters of the enzymatic reactions such as Vmax - the maximum rate of reaction and KM - the Michaelis constant. For experiments, the fluorogenic carbonate as a probe for a rapid determination of the kinetic parameters of hydrolases, such as lipases and esterases, was used. The microfluidic system together with the method described yields the kinetic constants calculated from the concentration of enzymatic product changes via a Michaelis-Menten model using the Lambert function W(x). This modular microfluidic system was validated on three selected enzymes (hydrolases).

Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion.

CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.

Rational Design of a Red-Emissive Fluorophore with AIE and ESIPT Characteristics and Its Application in Light-Up Sensing of Esterase.

The development of red fluorophores with efficient solid-state emission is still challenging. Herein, a red fluorophore 1 with aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics is rationally designed and facilely synthesized by attaching an electron-donor diethylamine and an electron-acceptor maleonitrile group to salicyladazine. In contrast to many red fluorophores which undergo serious aggregation-caused quenching (ACQ), compound 1 emits bright red fluorescence (λem = 650 nm, ΦF = 24.3%) in the solid state with a large Stokes shift of 174 nm. Interestingly, control compounds 2 and 3, which have similar structures as 1, exhibit obvious aggregation-caused quenching (ACQ) characteristics. The difference in the crystal structures of 1, 2, and 3 reveals that the interplanar spacing among molecules plays a decisive role in realizing the AIE characteristics of 1. Moreover, when the hydroxyl group of 1 was substituted by an esterase reactive acetoxyl, a fluorescence light-up probe 4 was developed for sensing of esterase based on the selective reaction between 4 and esterase to generate the AIE and ESIPT active molecule 1. The linear range for in vitro quantification of esterase is 0.01-0.15 U/mL with a detection limit of 0.005 U/mL. Probe 4 was also successfully applied to image esterase in mitochondria of living cells.

Pathogenic features of clinically significant coagulase-negative staphylococci in hospital and community infections in Benin.

In West Africa, very little consideration has been given to coagulase negative Staphylococci (CNS). Herein, we describe the features contributing to the pathogenicity of 99 clinically-significant independent CNS isolates associated with infections encountered at the National Teaching Hospital Center of Cotonou (Benin). The pathogenic potentials of nosocomial strains were compared with community strains. S. haemolyticus (44%), S. epidermidis (22%) and S. hominis (7%) were the most frequently isolated while bacteremia (66.7%) and urinary tract infections (24.2%) were the most commonly encountered infections. Most strains were resistant to multiple antibiotics, including penicillin (92%), fosfomycin (81%), methicillin (74%) and trimethoprim-sulfamethoxazole (72%). The most frequently isolated species were also the most frequently resistant to methicillin: S. hominis (100%), S. haemolyticus (93%) and S. epidermidis (67%). Screening of toxic functions or toxin presence revealed hemolytic potential in 25% of strains in over 50% of human erythrocytes in 1h. Twenty-six percent of strains exhibited protease activity with low (5%), moderate (10%) and high activity (11%), while 25% of strains displayed esterase activity. Three percent of strain supernatants were able to lyse 100% of human polymorphonuclear cells after 30min. Polymerase chain reaction and latex agglutination methods revealed staphylococcal enterotoxin C gene expression in 9% of S. epidermidis. A majority of hospital-associated CNS strains (68%) had at least one important virulence feature, compared with only 32% for community-acquired strains. The present investigation confirms that these microorganisms can be virulent, at least in some individual cases, possibly through genetic transfer from S. aureus.

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry.

Diagnostic possibilities from a serum sample-Clinical value of new methods within small animal reproduction, with focus on anti-Müllerian hormone.

During the last decade, analysis of anti-Müllerian hormone (AMH), highly conserved between mammalian species, has contributed to new information in reproductive endocrinology, due to clinically available diagnostic assays. AMH is produced solely in the gonads, in the Sertoli cells of testes and granulosa cells of the ovary, and thus offers possibilities to diagnose physiologic and pathologic conditions involving these organs. This article reviews indications for AMH analysis in cats and dogs, including diagnosing the presence of gonads, and granulosa or Sertoli cell tumours. Diagnostic challenges are addressed. One specific organ, the prostate, is commonly affected by pathologic changes in older dogs. A commercial assay for analysing canine prostatic specific esterase (CPSE) enables analysis of CPSE in clinical practice, of potential value in the workup of benign prostatic hyperplasia in male dogs. This is described in this review, as is a new method for analysis of steroids: liquid chromatography-tandem mass spectrometry LC-MS/MS. Steroids have since long been analysed in studies on reproduction, and LC-MS/MS has the advantage of allowing analysis of panels of multiple steroids from small sample volumes. Altogether, these available methods may give new insights into small animal reproduction and are valuable tools for the practicing veterinarian.

Polymer membrane ion-selective electrodes as a convenient tool for lipases and esterases assays.

We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates, and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored. To overcome those drawbacks we have proposed an assay based on potentiometry with ISEs for lipases and esterases activity determination. This electrochemical methodology represents an attractive tool for enzyme analysis, because of its low detection limit, independence from sample volume and from sample turbidity. The usefulness of this assay has been proven by the determination of the activity of various raw enzymes "acetone powders" isolated from animal tissues. Moreover, activities of fractions obtained during purification of one of those raw biocatalysts were also determined that way. The reliability of determination enzyme activity with ISE assay was proven by comparison with a classical spectrophotometric method.

Entomopoxvirus infection induces changes in both juvenile hormone and ecdysteroid levels in larval Mythimna separata.

Insect viruses are among the most important pathogens of lepidopteran insects. Virus-infected larvae often show developmental defects including a prolonged larval period and a failure to pupate, but the mechanisms by which insect viruses regulate host development need further investigation. In this study, the regulation of host endocrinology by a lepidopteran entomopoxvirus (EPV), Mythimna separata EPV (MySEV), was examined. When fourth instar M. separata were inoculated with MySEV occlusion bodies, pupation was prevented and the insects died during the final (sixth) larval instar. Liquid chromatography-MS analysis revealed that juvenile hormone (JH) titres in the haemolymph of MySEV-infected sixth instars were higher than those in mock-infected larvae. JH esterase (JHE) activity was also examined by kinetic assay using a colorimetric substrate. The level of JHE activity in the haemolymph of MySEV-infected larvae was generally lower than that found in mock-infected larvae. In contrast, ecdysteroid titre in the haemolymph of final-instar MySEV-infected larvae was lower than that found in mock-infected larvae when measured by radioimmunoassay. A statistically significant difference in the release of ecdysteroids from prothoracic glands (PGs) that were dissected from MySEV- or mock-infected sixth instar Day 3 larvae was not found following prothoracicotropic hormone (PTTH) exposure. Our results indicate that the release of ecdysteroids was reduced not by infection of the PGs by MySEV, but by reduced PTTH production from the brain. Taken together our study suggests that EPVs retard host development by both reducing ecdysone titre and maintaining status quo levels of JH by preventing its metabolism.

Cellulose-Based Biosensors for Esterase Detection.

Cellulose has emerged as an attractive substrate for the production of economical, disposable, point-of-care (POC) analytical devices. Development of novel methods of (bio)activation is central to broadening the application space of cellulosic materials. Ironically, such efforts are stymied by the inherent biocompatibility and recalcitrance of cellulose fibers. Here, we have elaborated a versatile, chemo-enzymatic approach to activate cellulosic materials for CuAAC "click chemistry", to develop new fluorogenic esterase sensors. Gentle, aqueous modification conditions facilitate broad applicability to cellulose papers, gauzes, and hydrogels. Tethering of the released fluorophore to the cellulose surface prevents signal degradation due to diffusion and enables straightforward, sensitive visualization with a simple light source in resource-limited situations.

Self-immolative versatile fluorogenic probes for screening of hydrolytic enzyme activity.

Enzyme triggered probes with a self-immolative linker for rapid and sensitive hydrolase detection through a cascade reaction have been reported. Their utility was proved by the preparation of three model compounds and their evaluation as enzyme substrates and demonstration of their applicability as fluorogenic probes for screening lipase, esterase and protease activities. These probes represent a new class of fluorogenic compounds, are stable under aqueous conditions and not susceptible to nonspecific degradation. The utilization of the carbamate cleavable linkage in a probe structure allows moving away of the bulky fluorophore from the enzyme recognition unit and targets different classes of enzymes with the same substrate.

Total esterase activity in human saliva: Validation of an automated assay, characterization and behaviour after physical stress.

Although saliva has esterase activity, this activity has not been characterized or studied in individuals subjected to physical stress. The aim of this report was to develop and validate an automated spectrophotometric assay for total esterase activity measurement in human saliva, as well as to study the contribution of different enzymes on this activity and its behaviour under physical stress in healthy subjects. The assay used 4-nitrophenyl acetate as substrate and was precise, accurate and provided low limits of detection and quantification. Inhibition with diisopropylfluorophosphate showed that cholinesterase, carboxylesterase and cholesterol esterase contributions not represented more than 20% of total esterase. Addition of standards of lipase and albumin to saliva samples showed that both proteins significantly contributed to esterase activity only when equal or higher than 11.6 IU/L and 250 µg/mL, respectively. Western blot analyses showed absence of paraoxonase-1 and high amount of carbonic anhydrase-VI. The high affinity of purified carbonic anhydrase-VI for the substrate supported a major contribution of this enzyme. Total esterase activity and alpha-amylase was measured in saliva samples from 12 healthy male students before and after participation in an indoor football match. The activity significantly increased after match and positively correlated with salivary alpha-amylase. This method could be used as a biomarker of physical stress in humans, with carbonic anhydrase-VI being the esterase that contributed more to the activity of the assay.

Far-red fluorogenic probes for esterase and lipase detection.

Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.