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Analytical Chemistry Apr 2015N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology...
N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.
Topics: Amino Acid Isomerases; Colorimetry; High-Throughput Screening Assays; Molecular Structure
PubMed: 25716802
DOI: 10.1021/ac5047328 -
Bioprocess and Biosystems Engineering Mar 2023Dye-contaminated wastewater discharge from textile and dye manufacturing industries is reported as a world worse water polluter due to the toxic and mutagenic behavior...
Dye-contaminated wastewater discharge from textile and dye manufacturing industries is reported as a world worse water polluter due to the toxic and mutagenic behavior of dyes. Peroxidase, one of the key enzymes of oxidoreductases, is widely distributed in nature and has been currently exploited in industries for various applications. Widespread applications of peroxidases are associated with their nonspecific nature towards a wide spectrum of substrates such as phenols, aromatic amines, pesticides, antibiotics, and synthetic dyes. The present study explored the potential of ammonium sulfate precipitated partially purified Brassica oleracea L. var. botrytis leaves peroxidase for degradation of reactive textile dyes Remazol Turquoise Blue 133 G and Drim Red CL4BN. Various physico-chemical parameters such as pH (2-9), temperature (20-70 ℃), enzyme activity (3-24 U/mL), concentrations of HO (0.4-1.4 Mm) and dye (10-100 mg/L) were optimized for enzymatic decolorization of both dyes' solution. Studies revealed that maximum degradation (95%) of Remazol Turquoise Blue 133 G with peroxidase was achieved with 25 mg/L of initial dye concentration, in the presence of 0.8 mM hydrogen peroxide with 45 min of incubation time, at pH 3, 4, and 5, and 70 °C. Maximal decolorization (97%) of Drim Red CL4BN was obtained at pH 2.0, in 10 min of incubation time at 45 ℃ using o-dianisidine hydrochloride as a redox mediator. In conclusion, the findings illustrate the prospect of Brassica oleracea peroxidase to remediate dye pollutants and dye-based industrial effluents in a green technology theme.
Topics: Peroxidase; Botrytis; Hydrogen Peroxide; Peroxidases; Coloring Agents; Textile Industry; Textiles; Plant Leaves; Brassica; Biodegradation, Environmental
PubMed: 36454313
DOI: 10.1007/s00449-022-02820-x -
Drug Metabolism and Personalized Therapy Sep 2015Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of...
BACKGROUND
Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of hemoglobin which is released to the serum is high; however, the amount of haptoglobin is not enough for binding all the released hemoglobins. Free hemoglobin has peroxidase activity (a pseudoenzyme) and has been indicated to be harmful for patients. This study is focused on the effect of cimetidine on peroxidase activity of hemoglobin.
METHODS
Erythrocytes were lysed to obtain hemoglobin. Peroxidase activity of hemoglobin was detected using o-dianisidine and H(2)O(2) as substrates.
RESULTS
Our results showed that the drug operated as an activator for the pseudoenzyme. Cimetidine bound to the pseudoperoxidase in an un-competitive manner and decreased the Km. Half maximal effective concentration (EC(50)) of cimetidine was determined to be about 12.5 mM. Alkaline pH increased the rate of reaction. Arrhenius plot showed that the activation energies of reactions in the absence and presence of drug were about 10.5 kJ/mol and 7.65 kJ/mol, respectively.
CONCLUSIONS
The results demonstrated that cimetidine activates the peroxidase activity of free hemoglobin. Hence, it is suggested that the prescription of cimetidine for the patients with hemolyses diseases may enhance the harmful effects of free hemoglobin in these patients.
Topics: Anti-Ulcer Agents; Biocatalysis; Cimetidine; Enzyme Activation; Hemoglobin A; Histamine H2 Antagonists; Humans; Hydrogen-Ion Concentration; Peroxidases; Temperature
PubMed: 26167985
DOI: 10.1515/dmpt-2014-0032 -
The Analyst Dec 2014A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose...
A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 μL of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 °C as long as four months.
Topics: Benzothiazoles; Biosensing Techniques; Blood Glucose; Colorimetry; Coloring Agents; Dianisidine; Enzymes, Immobilized; Equipment Design; Glucose Oxidase; Horseradish Peroxidase; Humans; Limit of Detection; Membranes, Artificial; Nanofibers; Reagent Strips; Sulfonic Acids
PubMed: 25343161
DOI: 10.1039/c4an01354a -
Pharmacological Research Dec 2015The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly...
The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and β-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.
Topics: Amino Acid Sequence; Animals; Biosimilar Pharmaceuticals; Computational Biology; Embryo, Nonmammalian; Epoetin Alfa; Erythropoietin; GATA1 Transcription Factor; Humans; Models, Animal; Molecular Sequence Data; Receptors, Erythropoietin; Recombinant Proteins; Sequence Alignment; Up-Regulation; Zebrafish
PubMed: 26361727
DOI: 10.1016/j.phrs.2015.09.004 -
Naunyn-Schmiedeberg's Archives of... May 2016Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory...
Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory bowel diseases. The aim of the present study was to characterize the role of melatonin receptors (MT) in the anti-inflammatory effect of melatonin and to assess the anti-inflammatory potential of two novel MT receptor agonists, Neu-P11 and Neu-P67, in the mouse model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Colitis was induced on day 1 by intracolonic (i.c.) administration of TNBS in 30 % ethanol in saline. Melatonin (4 mg/kg, per os (p.o.)), Neu-P11 (20 mg/kg, p.o.; 50 mg/kg, intraperitoneally (i.p.), 50 mg/kg, i.c.), and Neu-P67 (20 mg/kg, p.o.) were given twice daily for 3 days. Luzindole (5 mg/kg, i.p.) was injected 15 min prior to melatonin administration. On day 4, macroscopic and microscopic damage scores were assessed and myeloperoxidase (MPO) activity quantified using O-dianisidine-based assay. Melatonin significantly attenuated colitis in mice, as indicated by the macroscopic score (1.90 ± 0.34 vs. 3.82 ± 0.62 for melatonin- and TNBS-treated mice, respectively), ulcer score (0.87 ± 0.18 vs. 1.31 ± 0.19, respectively), and MPO activity (4.68 ± 0.70 vs.6.26 ± 0.94, respectively). Luzindole, a MT receptor antagonist, did not inhibit the anti-inflammatory effect of melatonin (macroscopic score 1.12 ± 0.22, ulcer score 0.50 ± 0.16); however, luzindole increased MPO activity (7.57 ± 1.05). MT receptor agonists Neu-P11 and Neu-P67 did not improve inflammation induced by TNBS. Melatonin, but not MT receptor agonists, exerts potent anti-inflammatory action in acute TNBS-induced colitis. Our data suggests that melatonin attenuates colitis by additional, MT receptor-independent pathways.
Topics: Animals; Anti-Inflammatory Agents; Colitis; Colon; Indoles; Male; Melatonin; Mice, Inbred BALB C; Peroxidase; Pyrans; Receptors, Melatonin; Trinitrobenzenesulfonic Acid
PubMed: 26899972
DOI: 10.1007/s00210-016-1214-x -
Chemistry, An Asian Journal Jun 2020In this work we have achieved epoxide to cyclic carbonate conversion using a metal-free polymeric catalyst under ambient CO pressure (1.02 atm) using a balloon setup....
In this work we have achieved epoxide to cyclic carbonate conversion using a metal-free polymeric catalyst under ambient CO pressure (1.02 atm) using a balloon setup. The triazine containing polymer (CYA-ANIS) was prepared from cyanuric chloride (CYA-Cl) and o-dianisidine (ANIS) in anhydrous DMF as solvent by refluxing under the N gas environment. The presence of triazine and amine functional groups in the polymer results in the adsorption of CO up to 7 cc/g at 273 K. This inspired us to utilize the polymer for the conversion of a series of functionalised epoxides into their corresponding cyclic carbonates in the presence of tetrabutyl ammonium iodide (TBAI) as co-catalyst. The product has wide range of applications like solvent in lithium ion battery, precursor for polycarbonate, etc. The catalyst was efficient for the conversion of different mono and di-epoxides into their corresponding cyclic carbonates under atmospheric pressure in the presence of TBAI as co-catalyst. The study indicates that epoxide attached with electron withdrawing groups (like, CH Cl, glycidyl ether, etc.) displayed better conversion compared to simple alkane chain attached epoxides. This is mainly due to the stabilization of electron rich intermediates produced during the reaction (e. g. epoxide ring opening or CO incorporation into the halo-alkoxide anion). This catalyst mixture was capable to maintain its reactivity up to five cycles without losing its activity. Post catalytic characterization clearly supports the heterogeneous and recyclable nature of the catalyst.
PubMed: 32270910
DOI: 10.1002/asia.201901277 -
ACS Chemical Biology May 2018X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding...
X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding and homohexameric protein organization. Three dimers interwoven in contact regions and tightened by hydrogen-bond-clamped guanidinium stacks along with regularly aligned water molecules form the basic structure of the enyzme. Intra- and intermolecular disulfide bridges further stabilize the enzyme preventing altogether the protein from denaturing up to a temperature of 90 °C, as evident from dynamic light scattering and the on-gel ortho-dianisidine assay. Every monomer binds one equivalent of orthovanadate in a cavity formed from side chains of three histidines, two arginines, one lysine, serine, and tryptophan. Protein binding occurs primarily through hydrogen bridges and superimposed by Coulomb attraction according to thermochemical model on density functional level of theory (B3LYP/6-311++G**). The strongest attractor is the arginine side chain mimic N-methylguanidinium, enhancing in positive cooperative manner hydrogen bridges toward weaker acceptors, such as residues from lysine and serine. Activating hydrogen peroxide occurs in the thermochemical model by side-on binding in orthovanadium peroxoic acid, oxidizing bromide with virtually no activation energy to hydrogen bonded hypobromous acid.
Topics: Binding Sites; Bromine; Density Functional Theory; Oxidation-Reduction; Peroxidases; Vanadates; X-Ray Diffraction
PubMed: 29665335
DOI: 10.1021/acschembio.8b00041 -
Biointerphases Mar 2017Mesoporous silica nanoparticles (MSNPs) have been used as an efficient and safe carrier for drug delivery and biocatalysis. The surface modification of MSNPs using...
Mesoporous silica nanoparticles (MSNPs) have been used as an efficient and safe carrier for drug delivery and biocatalysis. The surface modification of MSNPs using suitable reagents may provide a robust framework in which two or more components can be incorporated to give multifunctional capabilities (e.g., synthesis of noble metal nanoparticles within mesoporous architecture along with loading of a bioactive molecule). In this study, the authors reported on a new synthetic route for the synthesis of gold nanoparticles (AuNPs) within (1) unmodified MSNPs and (2) 3-trihydroxysilylpropyl methylphosphonate-modified MSNPs. A cationic polymer, polyethylenimine (PEI), and formaldehyde were used to mediate synthetic incorporation of AuNPs within MSNPs. The AuNPs incorporated within the mesoporous matrix were characterized by transmission electron microscopy, energy dispersive x-ray analysis, and high-resolution scanning electron microscopy. PEI in the presence of formaldehyde enabled synthetic incorporation of AuNPs in both unmodified and modified MSNPs. The use of unmodified MSNPs was associated with an increase in the polycrystalline structure of the AuNPs within the MSNPs. The AuNPs within modified MSNPs showed better catalytic activity than those within unmodified MSNPs. MSNPs with an average size of 200 nm and with a pore size of 4-6 nm were used for synthetic insertion of AuNPs. It was found that the PEI coating enabled AuNPs synthesis within the mesopores in the presence of formaldehyde or tetrahydrofuran hydroperoxide at a temperature between 10 and 25 °C or at 60 °C in the absence of organic reducing agents. The as-made AuNP-inserted MSNPs exhibited enhanced catalytic activity. For example, these materials enabled rapid catalytic oxidation of the o-dianisidine substrate to produce a colored solution in proportion to the amount of HO generated as a function of glucose oxidase-catalyzed oxidation of glucose; a linear concentration range from 80 to 800 μM and a detection limit as low as 80 μM were observed. The mesoscale pores of the as developed AuNP-inserted MSNPs were also used to entrap the hydrophobic drug paclitaxel. The results of this study indicate the potential use of the AuNP-inserted MSNPs in biocatalysis and drug delivery.
Topics: Biocatalysis; Drug Carriers; Formaldehyde; Gold; Hydrophobic and Hydrophilic Interactions; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Nanoparticles; Polyethyleneimine; Silicon Dioxide; Spectrometry, X-Ray Emission; Surface Properties; Temperature
PubMed: 28347142
DOI: 10.1116/1.4979200 -
Environmental Monitoring and Assessment Sep 2020Simple, low-cost, and sensitive methods for the assessment of hexavalent chromium as an important environmental pollutant are highly desirable, especially under...
Simple, low-cost, and sensitive methods for the assessment of hexavalent chromium as an important environmental pollutant are highly desirable, especially under resource-limited settings. Therefore, herein we propose an original approach for the simple, low-cost, selective, and extremely sensitive assessment of Cr(VI) utilizing its catalysis of the micellar sensitized o-dianisidine (DA)-hydrogen peroxide reaction. The initial rate of the amended reaction is monitored by tracing the oxidation product, either by a digital camera video recording or spectrophotometrically at 440 nm, for 120 s from mixing the reactants. The optimized reaction conditions were 5 mmol L DA, 0.6 mol L HO, 2.0 v/v% Tween 20, and 10 mmol L chloroacetate buffer (pH 4.5 ± 0.1), at 30 °C. The linear calibration graph extends to 90.0 ng mL Cr(VI) with detection limits (3S) of 0.8 and 1.0 ng mL, for the video recording and spectrophotometric procedures, respectively. The amended method was successfully applied to the assessment of Cr(IV) in natural and polluted industrial wastewaters. The analytical data were in excellent statistical harmony with those of the standard ETAAS method. The proposed method is two orders of magnitude more sensitive than the diphenylcarbazide standard spectrophotometric method.Graphical abstract.
Topics: Chromium; Colorimetry; Environmental Monitoring; Hydrogen Peroxide
PubMed: 32968831
DOI: 10.1007/s10661-020-08615-5