Hydrolysis Of P-nitrophenyl Phosphate Research Articles

Autoantibodies-Abzymes with Phosphatase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of MS (multiple sclerosis) evolution are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis simulating human MS) in C57BL/6 mice occurs due to the violation of bone marrow hematopoietic stem cell differentiation profiles, leading to the production of toxic for human autoantibody splitting MBP (myelin basic protein), MOG (mouse oligodendrocyte glycoprotein), five histones, DNA, and RNA. Here, we first analyzed the changes in the relative phosphatase activity of IgGs from C57BL/6 mice blood over time, corresponding to three stages of EAE: onset, acute, and remission. Antibodies have been shown to catalyze the hydrolysis of p-nitrophenyl phosphate at several optimal pH values, mainly in the range of 6.5-7.0 and 8.5-9.5. During the spontaneous development of EAE, the most optimal value is pH 6.5. At 50 days after the birth of mice, the phosphatase activity of IgGs at pH 8.8 is 1.6-fold higher than at pH 6.5. During spontaneous development of EAE from 50 to 100 days, an increase in phosphatase activity is observed at pH 6.5 but a decrease at pH 8.8. After mice were immunized with DNA-histone complex by 20 and 60 days, phosphatase activity increased respectively by 65.3 and 109.5 fold (pH 6.5) and 128.4 and 233.6 fold (pH 8.8). Treatment of mice with MOG at the acute phase of EAE development (20 days) leads to a maximal increase in the phosphatase activity of 117.6 fold (pH 6.5) and 494.7 fold (pH 8.8). The acceleration of EAE development after mice treatment with MOG and DNA-histone complex results in increased production of lymphocytes synthesizing antibodies with phosphatase activity. All data show that IgG phosphatase activity could be essential in EAE pathogenesis.

Indicators of Mineral Metabolism in the Oral Fluid in Patients with Gastroesophageal Reflux Disease

Disruption of the digestive system in the gastrointestinal tract, namely in gastroesophageal reflux disease, leads to the development of pathological processes in the oral cavity, changing the homeostasis of the viscoelastic gel layer of the esophageal mucosa. Thus, it has been proven that there are reflex connections between the receptor apparatus of the esophageal mucosa and the efferent nerve fibers of the salivary glands, which can be disrupted in GERD. Therefore, saliva is involved in providing effective protection of the esophagus. As a result, the content of mineral components changes significantly: in particular, the level of calcium and enzyme composition.
 Aim to study the indicators of mineral metabolism: the content of calcium, alkaline, and acid phosphatases in mixed saliva in patients with gastroesophageal reflux disease.
 Methods 60 patients of various ages who underwent inpatient treatment at the IFNMU University Clinic were examined, where pH-metry was performed to determine acidity. The main group consisted of patients diagnosed with GERD, with decreased and increased acidity. The content of indicators of mineral metabolism in saliva was determined as follows: calcium (Ca) - photometric method with arsenase -111; acid phosphatase (AC) was determined by the Hillman method, alkaline phosphatase (AL) by hydrolysis of p-nitrophenyl phosphate at pH -10.4, release of p-nitrophenol and phosphate. The norm of indicators of mineral metabolism was established on 30 practically healthy volunteers. Statistical processing of data was carried out using the Microsoft Excel program by calculating the arithmetic mean.
 Conclusions thus, the results obtained indicate that in GERD there is a violation of mineral metabolism in the oral fluid. The oral fluid is the first to come into contact with the reflux agent. This leads to a violation of saliva neutralization, so the study of saliva mineral composition can be considered as an early diagnostic marker for gastroesophageal reflux disease.

Matrix redox interference-free nanozyme-amplified detection of Hg2+ using thiol-modified phosphatase-mimetic nanoceria

Given heavy metals pose huge threats to ecological system and human health, it is significant to monitor them. With the favorable feature of catalytic signal amplification, redox-type nanozymes have been widely used to detect heavy metals. However, the presence of redox substances in samples can interfere with these catalytic reactions, impacting the accuracy and repeatability of detection. To avoid the situation, here we designed a non-redox nanozyme (3-mercaptopropionic acid modified nanoceria, MPA-CeO2) and developed a novel approach free from matrix redox interference to detect Hg2+. The well-dispersed MPA-CeO2 showed high and stable phosphatase-mimetic activity to catalyze the hydrolysis of colorless p-nitrophenyl phosphate (p-NPP) to yellow p-nitrophenol (p-NP). The addition of Hg2+ could bind specifically onto the MPA-CeO2 particles through S–Hg bond and cause the latter to aggregate, thereby suppressing the ability of MPA-CeO2 with shielded active surfaces to hydrolyze p-NPP. According to the simple principle, highly sensitive and specific colorimetric measurement of Hg2+ was achieved, and excellent reliability and practicability were demonstrated by real sample analysis. To our best knowledge, this study is the first one of detecting Hg2+ using phosphatase-mimicking nanozymes, and it will inspire the exploration of new analytical methods via designing non-redox nanozymes for various targets.

Designed Metal-Containing Peptoid Membranes as Enzyme Mimetics for Catalytic Organophosphate Degradation.

The detoxification of lethal organophosphate (OP) residues in the environment is crucial to prevent human exposure and protect modern society. Despite serving as excellent catalysts for OP degradation, natural enzymes require costly preparation and readily deactivate upon exposure to environmental conditions. Herein, we designed and prepared a series of phosphotriesterase mimics based on stable, self-assembled peptoid membranes to overcome these limitations of the enzymes and effectively catalyze the hydrolysis of dimethyl p-nitrophenyl phosphate (DMNP)─a nerve agent simulant. By covalently attaching metal-binding ligands to peptoid N-termini, we attained enzyme mimetics in the form of surface-functionalized crystalline nanomembranes. These nanomembranes display a precisely controlled arrangement of coordinated metal ions, which resemble the active sites found in phosphotriesterases to promote DMNP hydrolysis. Moreover, using these highly programmable peptoid nanomembranes allows for tuning the local chemical environment of the coordinated metal ion to achieve enhanced hydrolysis activity. Among the crystalline membranes that are active for DMNP degradation, those assembled from peptoids containing bis-quinoline ligands with an adjacent phenyl side chain showed the highest hydrolytic activity with a 219-fold rate acceleration over the background, demonstrating the important role of the hydrophobic environment in proximity to the active sites. Furthermore, these membranes exhibited remarkable stability and were able to retain their catalytic activity after heating to 60 °C and after multiple uses. This work provides insights into the principal features to construct a new class of biomimetic materials with high catalytic efficiency, cost-effectiveness, and reusability applied in nerve agent detoxification.

Novel dual-emission sulfur quantum dot sensing platform for quantitative monitoring of pesticide 2,4-dichlorophenoxyacetic acid

In this work, a novel environment-friendly dual-emission Rhodamine B modified sulfur quantum dots (RhB-SQDs) sensing platform was established to economically monitor organochlorine pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) through regulating the activity of alkaline phosphatase (ALP). This dual emission RhB-SQDs exhibited excellent fluorescence and high photostability with emission wavelengths of 455 nm and 580 nm. ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate to p-nitrophenol, which quenched RhB-SQDs fluorescence at 455 nm due to the internal filtration effect, but had no effect the fluorescence intensity of RhB-SQDs at 580 nm. When 2,4-D was present, the activity of ALP was specifically inhibited and enzymatic reaction was interrupted, leading to the reduction of p-nitrophenol production, so the fluorescence of RhB-SQDs at 455 nm was restored. It demonstrated a good linear relationship between the concentration of 2,4-D and F455/F580 in the range of 0.050–0.500 μg mL−1, with a detection limit of 17.3 ng mL−1. The dual-emission fluorescent probe was successfully realized in the identification of 2,4-D in natural water samples and vegetables with the advantages of exceptional accuracy, immunity to interference, and selectivity. The platform offers a fresh look at pesticide monitoring and has the potential to prevent pesticide-related health issues.

Ultrasensitive inner filter effect fluorescence sensing platform for alkaline phosphatase based on arginine surface-engineered gold nanoclusters

Developing a simple yet powerful method for enzyme activity monitoring is of great significance in the early diagnosis of diseases. Herein, we designed a precise spectral overlap-triggered inner filter effect (IFE) fluorescence sensing platform for alkaline phosphatase (ALP) detection. ALP could catalyze hydrolysis of p-nitrophenylphosphate (PNPP) to generate p-nitrophenol (PNP) and PNP acts as a powerful acceptor in IFE reaction system to influence the excitation of fluorescent donors. Based on this principle, a standardized procedure was proposed for rational screening of high-performance donor/acceptor pairs to maximize IFE efficiency. The results proved that perfect spectral overlap and high quantum yield of fluorescent donors synergistically contribute to maximizing IFE efficiency. In this case, a high quantum yield of 51.7% of the arginine surface-engineered gold nanoclusters (Arg/ATT-AuNCs) produced perfect spectral overlap with PNP and achieved maximal IFE efficiency, which was eventually selected as fluorescent signal tag for ALP detection. The Arg/ATT-AuNCs-based approach achieved ultrasensitive detection of ALP with a detection limit of 0.0062 U/L, which exhibited 4.2–25.8-fold improvement compared to other donors. Furthermore, the accuracy and reliability of established method in real human serum samples were verified by spiking analysis and commercial kits, thus providing a promising platform for monitoring ALP. This work aims to provide a standardized procedure for rational screening high-performance donor/acceptor pairs and to develop an ultrasensitive IFE sensing platform for biomarkers detection, which will open up a promising horizon for other biomarkers detection in clinical diagnostics.

Tetranuclear iron(III) complexes with a carboxylate-rich ligand as synthetic mimics of phosphoesterases in aqueous media

The characterization and catalytic activities of two tetranuclear Fe(III) complexes of a carboxylate-rich ligand, N,N’-Bis[2-carboxybenzomethyl]-N,N’ -Bis[carboxymethyl]-1,3-diaminopropan-2-ol (H5ccdp) ligand towards the hydrolysis of p-nitrophenyl phosphate (PNPP) and bis(p-nitrophenyl) phosphate (BNPP) substrates in aqueous systems are described. Kinetic investigations were carried out using UV–vis spectrophotometric techniques at 25 °C and 37 °C and varied pH conditions (7 – 9.5). The kinetic studies showed that the turnover rate (kcat) values for the hydrolysis of PNPP by [Fe4(ccdp)2(μ-O)(μ-OH)(d-GluA)2]3- were marginally higher than [Fe4(ccdp)2(OH)2(o-phth)2]4- at 9.24 × 10-6 s−1 (pH 8 and 37 °C) and 4.27 × 10-7 s−1 (pH 8 and 25 °C), respectively. However, similar comparisons among the BNPP hydrolysis showed the turnover rate values for [Fe4(ccdp)2(OH)2(o-phth)2]4- was marginally better than [Fe4(ccdp)2(μ -O)(μ –OH)(d-GluA)2]3- with values of 1.85 × 10-6 s−1 (pH 8 and 37 °C) and 6.23 × 10-7 (pH 8 and 37 °C). ESI-MS techniques detected catalyst-substrate adduct species for both metal complexes with PNPP. Additionally, both complexes also showed evidence for complex-substrate-nucleophile adduct formation with both BNPP and PNPP substrates via ESI-MS. Based on the kinetic activity and structural information obtained in this study, a reaction mechanism for the hydrolysis of phosphorester has been proposed.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01–10.0 and 50–300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.Electronic Supplementary MaterialSupplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV—vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

Construction and analysis of a food-grade Lactiplantibacillus plantarum esterase/lipase overexpression system

Lactiplantibacillus plantarum (L. plantarum) is widely used in the food industry, especially in meat fermentation and dairy processing, due to its lipase and esterase activities. In this study, lp_1002 from L. plantarum WCFS1 was cloned into the food-grade gene expression vector pMG36n, with nisin used as a selective marker. Furthermore, pMG36n- lp_1002 was retransformed into L. plantarum WCFS1, and lp_1002 was overexpressed in the newly constructed L. plantarum WCFS1 strain. Moreover, recombinant Lp_1002 showed the characteristics of both lipase and esterase. The results from hydrolysis of p-nitrophenylphosphate (pNPP) and triglycerides showed that the hydrolysis activities of the recombinant Lp_1002 were 420% and 360% higher than that of the original strain, respectively. In addition, the results of wine MLF fermentation suggest that the recombinant strain significantly reduced the contents of ethyl esters, acetate esters and other types of esters, which affected wine aroma during brewing. Therefore, the recombinant strain has great application potential in food fermentation.

A label-free and modification-free ratiometric electrochemical strategy for enhanced natural enzyme detection using a bare electrode and nanozymes system.

Ratiometric electrochemical assays have been demonstrated to be more sensitive and selective in various sensing events, mainly due to their affordable built-in correction and good self-reference capability. But it is known that complicated modification and labeling operations usually are necessary for the construction of ratiometric electrochemical assays, therefore is a hot and important issue needing consideration carefully. We herein report a new yet simple bare electrode-based ratiometric electrochemical bioassay to achieve sensitive and selective analysis of alkaline phosphatase (ALP), using a liquid phase system that contains CoOOH nanozymes and commercially available indicator substrate. This proposed bioassay works based on the ratiometric change of dual electrochemical signals, arising from an exclusive target ALP-triggered hydrolysis of electrochemical substrate p-nitrophenyl phosphate (PNPP). In this design, the two hydrolyzed products of electrochemically active p-nitrophenol (PNP) and electrochemically inactive phosphate anion (PO43-) are responsible together for the ratiometric electrochemical analysis of ALP. PNP exhibits a straightforward current response toward ALP content; however, PO43- cannot show a direct electrochemical signal thus is rationally designed to offer an alternative response by linking it with the specific CoOOH nanozyme-catalyzed reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, in which the nanozyme-catalyzed product oxTMB shows a direct reduction current at the GCE, and significantly decreases with increasing PO43- species due to the good inhibition of PO43- toward CoOOH nanozyme activity. As a result, a ratiometric electrochemical strategy for ALP analysis with a low limit of detection of 0.366 U/L (S/N = 3) was successfully achieved by integrating the above direct and indirect dual electrochemical responses. This developed bioassay can allow the quantitative diagnosis of ALP activity especially with a label-free and modification-free merit, therefore paving the way for simple, convenient, and portable electroanalytical tools in biosensing design and application.

Homodinuclear copper(II) and zinc(II) complexes of a carboxylate-rich ligand as synthetic mimics of phosphoester hydrolase in aqueous solutions

The synthesis, characterization and catalytic activities of two homodinuclear Cu(II) and Zn(II) complexes of a carboxylate-rich ligand, N,N′-Bis[2-carboxybenzomethyl]-N,N′ -Bis[carboxymethyl]-1,3-diaminopropan-2-ol (H5ccdp) ligand towards the hydrolysis of (p-nitrophenyl phosphate) (PNPP) and bis(p-nitrophenyl) phosphate (BNPP) substrates in aqueous systems are described. Kinetic investigations were carried out using UV–Vis spectrophotometric techniques at 25 °C and 37 °C and different pH (7–10) conditions. The kinetic studies revealed that the turnover rate (kcat) values among the PNPP hydrolysis systems, the highest and the lowest kcat values were displayed by [Cu2(ccdp)(μ-OAc)]2− at 2.34 × 10−6 s−1 (pH 8 and 37 °C) and 2.13 × 10−8 s−1 (pH 8 and 25 °C), respectively. However, similar comparisons among the BNPP hydrolysis revealed that highest and the lowest kcat values were displayed by [Zn2(ccdp)(μ-OAc)]2− at 4.64 × 10−8 s−1 (pH 9 and 37 °C) and 2.38 × 10−9 (pH 9 and 25 °C). Significantly enough, the catalyst−substrate adduct species containing a metal bound PNPP and BNPP have been detected by ESI-MS techniques. Additionally, a PNPP-bound copper complex has been isolated and crystalized using single crystal X-ray diffraction technique. Based on the structural and activity information obtained in this study, reaction mechanisms for the hydrolysis of PNPP have been proposed.

Enhanced catalytic stability of acid phosphatase immobilized in the mesospaces of a SiO2-nanoparticles assembly for catalytic hydrolysis of organophosphates

Abstract Acid phosphatase (APase) was immobilized in discrete mesospaces of an assembly of silica nanoparticles (SiO2NPA) to be used as a heterogeneous catalyst for the hydrolysis of organophosphates often found as pesticide residues in agricultural products. APase immobilized in SiO2NPA exhibited higher catalytic activity than APase supported on conventional porous supports for the hydrolysis of p-nitrophenyl phosphate (p-NPP) with quantitative yield of p-nitrophenol below pH 5.5 due to the efficient diffusion of substrates in the SiO2NPA. Especially, a heterogeneous catalyst prepared by the co-accumulation method, in which silica nanoparticles (SiO2NPs) dispersion containing APase was simply dried to assemble a composite catalyst (APase/SiO2NPA), exhibited four times faster rate for the hydrolysis of p-NPP than the catalyst prepared by the equilibrium adsorption of APase in pre-assembled SiO2NPA. The catalytic stability of immobilized APase above pH 6.0 was enhanced by surface modification of SiO2NPs with 3-aminopropyltriethoxysilane (APase/SiO2NPA-NH2) due to the strong electrostatic interaction between APase and the protonated amino groups at the pH condition. Such stability enhancement was hardly obtained by cross-linking treatment of SiO2NPA to improve the robustness. These results suggest that electrostatic interaction between APase and SiO2NPs is crucial to enhance catalytic stability in the wide range of pH as well as preparation methods for the stable encapsulation.

Ratiometric fluorescence determination of alkaline phosphatase activity based on dual emission of bovine serum albumin-stabilized gold nanoclusters and the inner filter effect.

A novel and convenient method for the ratiometric fluorescence detection of alkaline phosphatase (ALP) activity was proposed based on dual emission of bovine serum albumin-templated gold nanoclusters (BSA-AuNCs) and the mechanism of the inner filter effect between BSA-AuNCs and p-nitrophenol (PNP). First, ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate (PNPP) to produce PNP. PNP effectively quenched the emission peak of BSA-AuNCs at 410 nm because of the overlap in absorbance feature of PNP and the fluorescence spectrum of BSA-AuNCs, and the peak at 650 nm was almost unaffected. Thus, a sensitive ratiometric method for detection of ALP activity was developed using the fluorescence intensity of BSA-AuNCs at 650 nm as a reference signal. ALP activity versus the ratio of fluorescence intensities at 410 and 650 nm showed good linearity between 0.2 and 5 mU mL-1 (R2 = 0.9931) and high sensitivity with a detection limit of 0.03 mU mL-1 (S/N = 3). The developed sensing method was successfully applied to investigate ALP inhibitors and detect ALP in serum samples.

Anti-inflammatory activity of high and low methoxylated apple pectins, in vivo and in vitro

One of the possible mechanisms of the anti-inflammatory action of pectins is associated with the inhibition of excessive pro-inflammatory activity of macrophages - the cells that regulate inflammation intensity and reparative regeneration. It has been found that pectins with a low degree of methyl esterification of the carboxyl groups of the galacturonan core of the macromolecule exhibit this effect. In addition to leukocytes, intestinal epithelial cells are also involved in the pathogenesis of inflammatory bowel diseases. However, to date, there have been insufficient studies of the effect of pectin methyl esterification on the inflammatory response of enterocytes. The aim of the research was to evaluate the effect of the degree of pectin methyl esterification on inflammation of the colon in mice after oral administration and on the inflammatory response of human colon epithelium cells of the Caco-2 line in vitro. Material and methods. In a prospective study, 40 male BALB/c mice weighing 20-25 g were used, 10 animals in each group. Solutions of apple pectins (200 mg/0.2 ml) were orally administered to mice through a plastic catheter 24 h before the induction of colitis. The control mice received water, and prednisone administration at a dose of 5 mg/kg of body weight was used as a positive control. Colon inflammation in mice was induced by a single rectal administration of 5% acetic acid (0.1 ml). A day later, the degree and area of the lesion was assessed using a light microscope, the activity of myeloperoxidase in the wall of the colon was determined by spectrophotometry. The effect of pectins on metabolic activity, intercellular permeability, Tumor Necrosis Factor α generation and alkaline phosphatase (ALP) activity in Caco-2 cells was assessed. Results. It was found that low-methyl esterified pectin AU701, which contains more than 70% of free carboxyl groups, inhibited colon inflammation in mice. High methyletherified pectin AU201, in which more than 70% of the carboxyl groups are replaced by methyl ester, didn't affect inflammation. It was revealed that pretreatment of Caco-2 cells with AU701 and AU201 pectins prevented lipopolysaccharide-induced increase in intercellular permeability and reduced the pro-inflammatory response of Caco-2 cells to LPS. After incubation of Caco-2 cells with AU701 pectin, the rate of hydrolysis of p-nitrophenyl phosphate (an alkaline phosphate substrate) increased by 40%. Pectin AU201 had no effect on the alkaline phosphatase activity of enterocytes. Conclusion. Thus, it was found that low-methyl esterified pectin AU701 inhibits inflammation both in vivo and in vitro. High-methyl esterified pectin AU201 suppresses pro-inflammatory reactions only in vitro. The ability of pectins to inhibit intestinal inflammation has a multifactorial nature, and is due, inter alia, to their ability to stimulate the expression of alkaline phosphatase by enterocytes.

Expression and physicochemical characterization of an N-terminal polyhistidine-tagged phosphotriesterase from the soil bacterium Brevundimonas diminuta

Abstract In this study, a truncated organophosphate-degrading (opd) gene encoding a Brevundimonas diminuta phosphotriesterase (BdPTE) without its leader peptide was artificially synthesized and cloned into the expression vector pQE-30 to yield pQE-BdPTE. The His6-tagged 37.8-kDa enzyme was purified from the cell-free extract of isopropyl thio-β- d -galactoside (IPTG)-induced E. coli M15 (pRep4) cells harboring pQE-BdPTE to near homogeneity by metal chelate affinity chromatography. The assay conditions for His6-tagged BdPTE activity were optimized at 60 °C, pH 8.0, and 1.0 mM Co2+ ion. Kinetic analysis of His6-tagged BdPTE-catalyzed hydrolysis of diethyl p-nitrophenyl phosphate (paraoxon-ethyl) showed that its Km and kcat values were 21.9 ± 1.4 μM and 311.6 ± 12.3 s−1, respectively. Also, the recombinant enzyme had the ability to efficiently degrade the two most commonly used OP pesticides, ethion and fenthion, in Taiwan. His6-tagged BdPTE with 1 mM Co2+ was very stable during the storage period of 35 days at 4 °C. Urea- and guanidine hydrochloride (GdnHCl)-induced denaturation studies showed that the purified enzyme had [denaturant]0.5,N–U values of 6.7 and 3.4 M, respectively. Notably, His6-tagged BdPTE did exhibit some degree of tolerance to a variety of water-miscible organic co-solvents. Dimethyl sulfoxide (DMSO), acetone, dimethylformamide (DMF), and ethanol were obviously harmful to the hydrolytic activity of the enzyme at concentrations above 50% (v/v), while acetonitrile was the highest tolerated co-solvent. The organic solvent-tolerant feature verifies His6-tagged BdPTE highly suitable as a remediation agent for the biodegradation of non-polar OP pesticides.

In Situ Formation of 2,3-Diaminophenazine for Evaluation of Alkaline Phosphatase Activity via the Inner Filter Effect

We report a fluorescence assay for alkaline phosphatase (ALP) detection using in situ generation of 2,3-diaminophenazine (OPDox) through an inner filter effect (IFE). AgNO3 can oxidize o-phenylenediamine in a short time to obtain fluorescent OPDox. p-nitrophenol is obtained from ALP-catalyzed hydrolysis of p-nitrophenylphosphate, which can result in the fluorescence quenching of OPDox via the IFE. Consequently, in situ generation of OPDox can be utilized for evaluating the activity of ALP. The fluorescence of OPDox decreases linearly with increasing concentration of ALP in the range from 0.1 to 8.0 mU mL-1, and the detection limit is calculated to be 0.05 mU mL-1. More importantly, this assay has been used to construct a logic gate. The IFE-based fluorescence assay exhibits several distinctive advantages, including high sensitivity, good selectivity, and simple operation. Consequently, it may pave the way for the detection of ALP by utilization of the in situ generation of fluorophores.

Role of metal cations and oxyanions in the regulation of protein arginine phosphatase activity of YwlE from Bacillus subtilis

Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modification. Protein arginine phosphatase YwlE negatively regulates arginine phosphorylation and consequently induces the expression of stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus virulence. However, little is known about the factors that affect the enzymatic activity of YwlE with the exception of the effect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by YwlE, we investigate the role of metal cations and oxyanions in the regulation of YwlE activity. Interestingly, among the various cations that we tested, Ca2+ activates YwlE, while other cations, including Ag+, Co2+, Cd2+, and Zn2+, are inhibitory. Furthermore, as chemical analogues of phosphate, oxyanions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates YwlE activity. Specifically, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H2O2, resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), β-me (β-mercaptoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance YwlE activity. Additionally, as a stable analogue to pArg, pAIE binds to YwlE with a Kd of 149.1 nM and a binding stoichiometry n of 1.2 and inhibits YwlE with an IC50 of 316.3 ± 12.73 μM. The inhibition and activation of YwlE may have broad implications for the physiology, pharmacology and toxicology of metal cations and oxyanions.

Upconversional Nanoprobes with Highly Efficient Energy Transfer for Ultrasensitive Detection of Alkaline Phosphatase.

Sensitive detection of alkaline phosphatase (ALP) activity in human serum is important for diagnosis of various diseases. In this work, a novel sandwich-structured upconversion nanoparticle, NaYF4:Yb/Er@NaErF4@NaYF4, is fabricated to construct an upconversional nanoprobe for ultrasensitive detection of phosphate and ALP activity. The inner shell of NaErF4 bridges the emitters in the core with the external luminescence quenchers to greatly improve the energy transfer efficiency. The quencher, herein, is a coordination complex formed between sulfosalicylic acid and ferric ions. Owing to the higher affinity for phosphate, ferric ions dissociate from the complex and potently combine with phosphate ions, thus interrupting the energy transfer process and recovering the luminescence. This upconversional nanoprobe shows rapid and sensitive detection of phosphate with a limit of detection of 2.5 nM. Because ALP catalyzes the hydrolysis of p-nitrophenyl phosphate to form p-nitrophenol and inorganic phosphate ions, the nanoprobe is further utilized to achieve sensitive detection of ALP with a limit of detection of 0.5 μU/mL. This novel strategy offers a new opportunity for developing sensitive upconversional nanoprobes and many other energy transfer-based applications.

Recyclable metal nanoparticle-immobilized polymer dot on montmorillonite for alkaline phosphatase-based colorimetric sensor with photothermal ablation of Bacteria

Development of simultaneous bacteria detection and eradication with simple, rapid, and reusable material is important in addressing bacterial contamination issues. In this study, we utilized the expression of alkaline phosphatase (ALP) from bacteria to design fluorescence ON/OFF system for bacteria detection, also using metal oxide nanoparticle for obtaining antibacterial activity and recyclability. The fluorescent-based biosensor with antibacterial activity was prepared by intercalating ALP-sensitive polymer dot (PD) containing β-cyclodextrin (β-CD) onto montmorillonite (MMT) as loading matrix via ionic exchange reaction, followed by immobilization of magnetic iron oxide (Fe3O4) and NIR-responsive cesium tungsten oxide (CsWO3). The PD-βCD-MMT/Fe3O4–CsWO3 nanocomposite exhibited strong fluorescence intensity, which was quenched in the presence of bacterial ALP (0–1000 U/L) due to hydrolysis of p-nitrophenyl phosphate (NPP) into p-nitrophenol (NP) in the hydrophobic site of β-CD. Furthermore, the nanocomposite could detect both gram-negative Escherichia coli and gram-positive Staphylococcus aureus in the range of 101–107 CFU/mL (LOD 5.09 and 4.62 CFU/mL, respectively), and showed high antibacterial activity against bacteria by generating photothermal heat under 5 min NIR irradiation, causing damage to bacterial cells. This material also demonstrated recyclability via magnetic field exposure due to the presence of Fe3O4. In addition, the fluorescence can be recovered following pH shock and re-conjugation of β-CD molecules. After 4 cycles, nanocomposite still showed stable photothermal effects and fluorescence-based bacteria detection. Thus, this reusable material offers promising approach for simultaneous bacteria detection and killing, which is simple, rapid, and effective.

Consequences of Heterogeneous Crowding on an Enzymatic Reaction: A Residence Time Monte Carlo Approach.

Translational diffusion of a free substrate in crowded metabolically active spaces such as cell cytoplasm or mitochondrial matrix is punctuated by collisions and nonspecific interactions with soluble/immobile macromolecules/macrostructures in a variety of shapes/sizes. It is not understood how such disruptions alter enzyme reaction kinetics in such spaces. A novel Monte Carlo (MC) technique, “residence time MC”, has been developed to study the kinetics of a simple enzyme–substrate reaction in a crowded milieu using a single immobile enzyme in the midst of diffusing substrates and products. The reaction time lost while the substrate nonspecifically interacts or is transiently trapped with ambient macromolecules is quantified by introducing the residence time “tau”. Tau scales with the size of crowding macromolecules but makes the knowledge of their shape redundant. The residence time thus presents a convenient parameter to realistically mimic the sticky surroundings encountered by a diffusing substrate in heterogeneously crowded physiological spaces. Results reveal that for identical substrate concentration and excluded volume, increase in tau significantly diminished enzymatic product yield and reaction rate, slowed down substrate/product diffusion, and prolonged their relaxation times. A smooth transition from the anomalous subdiffusive motion to normal diffusion at long time limits was observed irrespective of the value of tau. The predictions from the model are shown to be in qualitative agreement with in vitro experimental data revealing the rate of alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate in the midst of 40/500/2000 kDa dextrans. Our findings from the residence time MC model also attempt to rationalize previously unexplained experimental observations in crowded enzyme kinetics literature. Furthermore, major insights to emerge from this study are the reasons why free diffusion of the substrate in crowded physiological spaces is detrimental to enzyme function. It is argued that organized enzyme clusters such as “metabolon” may perhaps exist to regulate the substrate translocation in such sticky physiological spaces to maintain optimal enzyme function. In summary, this work provides key insights explaining why absence of substrate channeling can dramatically slow down enzyme reaction rate in crowded metabolically active spaces.