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International Journal of Molecular... Feb 2019Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however,...
Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing -glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four -glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2'-azino-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.
Topics: Catalysis; Enzyme Activation; Enzyme Stability; Escherichia coli; Glycosylation; Horseradish Peroxidase; Kinetics; Mutagenesis, Site-Directed; Recombinant Proteins; Temperature
PubMed: 30791559
DOI: 10.3390/ijms20040916 -
Biomacromolecules May 2022Thin films of cellulose ferulate were designed to study the formation of dehydrogenation polymers (DHPs) on anchor groups of the surface. Trimethylsilyl (TMS) cellulose...
Thin films of cellulose ferulate were designed to study the formation of dehydrogenation polymers (DHPs) on anchor groups of the surface. Trimethylsilyl (TMS) cellulose ferulate with degree of substitution values of 0.35 (ferulate) and 2.53 (TMS) was synthesized by sophisticated polysaccharide chemistry applying the Mitsunobu reaction. The biopolymer derivative was spin-coated into thin films to yield ferulate moieties on a smooth cellulose surface. Dehydrogenative polymerization of coniferyl alcohol was performed in a Quartz crystal microbalance with a dissipation monitoring device in the presence of HO and adsorbed horseradish peroxidase. The amount of DHP formed on the surface was found to be independent of the base layer thickness from 14 to 75 nm. Pyrolysis-GC-MS measurements of the DHP revealed β-O-4 and β-5 linkages. Mimicking lignification of plant cell walls on highly defined model films enables reproducible investigations of structure-property relationships.
Topics: Cellulose; Horseradish Peroxidase; Hydrogen Peroxide; Lignin; Polymerization
PubMed: 35438964
DOI: 10.1021/acs.biomac.2c00096 -
International Journal of Molecular... Apr 2023Phenols are widely used in industries despite their toxicity, which requires governments to limit their concentration in water to 5 mg/L before discharge to the city...
Phenols are widely used in industries despite their toxicity, which requires governments to limit their concentration in water to 5 mg/L before discharge to the city sewer. Thus, it is essential to develop a rapid, simple, and low-cost detection method for phenol. This study explored two pathways of peroxidase immobilization to develop a phenol detection system: peroxidase encapsulation into polyelectrolyte microcapsules and peroxidase captured by CaCO. The encapsulation of peroxidase decreased enzyme activity by 96%; thus, this method cannot be used for detection systems. The capturing process of peroxidase by CaCO microspherulites did not affect the maximum reaction rate and the Michaelis constant of peroxidase. The native peroxidase-Vmax = 109 µM/min, Km = 994 µM; CaCO-peroxidase-Vmax = 93.5 µM/min, Km = 956 µM. Ultimately, a reusable phenol detection system based on CaCO microparticles with immobilized peroxidase was developed, capable of detecting phenol in the range of 700 ng/mL to 14 µg/mL, with an error not exceeding 5%, and having a relatively low cost and production time. The efficiency of the system was confirmed by determining the content of phenol in a paintwork product.
Topics: Phenol; Peroxidase; Phenols; Peroxidases; Enzymes, Immobilized; Horseradish Peroxidase
PubMed: 37047739
DOI: 10.3390/ijms24076766 -
The Journal of Biological Chemistry Dec 1985Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish...
Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide-glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound.
Topics: Carbon Radioisotopes; Diethylstilbestrol; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Horseradish Peroxidase; Oxygen Consumption; Peroxidases; Prostaglandin-Endoperoxide Synthases
PubMed: 2999150
DOI: No ID Found -
Biomacromolecules Mar 2022The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed cross-linking of proteins remains a useful strategy to modulate hydrogel formation. Here,...
The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed cross-linking of proteins remains a useful strategy to modulate hydrogel formation. Here, we demonstrate that the presence of positively charged lysines in silk-elastin-like polymers impacts the thermal transition temperature of these proteins, while the location in the primary sequence modulates the reactivity of the tyrosines. The positively charged lysine side chains decreased π-π interactions among the tyrosines and reduced the rate of formation and number of HRP-mediated dityrosine bonds, dependent on the proximity of the charged group to the tyrosine. The results suggest that the location of repulsive charges can be used to tailor the reaction kinetics for enzymatic cross-linking, providing further control of gelation rates for gel formation and the resulting protein-based gel characteristics.
Topics: Cross-Linking Reagents; Elastin; Horseradish Peroxidase; Hydrogels; Silk; Tyrosine
PubMed: 35113522
DOI: 10.1021/acs.biomac.1c01192 -
Biosensors Jan 2022Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological...
Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L, 0.9831), low limit of detection (0.082 nmol L), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.
Topics: Colorimetry; Gold; Horseradish Peroxidase; Human Growth Hormone; Humans; Immunoassay
PubMed: 35200326
DOI: 10.3390/bios12020065 -
Molecules (Basel, Switzerland) Oct 2019Acting as a "green" manufacturing route, the enzyme toolbox made up of galactose oxidase, catalase, and horseradish peroxidase can achieve a satisfactory yield of...
Acting as a "green" manufacturing route, the enzyme toolbox made up of galactose oxidase, catalase, and horseradish peroxidase can achieve a satisfactory yield of 2,5-diformylfuran derived from 30 mM hydroxymethylfurfural. However, as the concentration of hydroxymethylfurfural increases, the substrate causes oxidative damage to the activity of the tri-enzyme system, and the accumulated hydrogen peroxide produced by galactose oxidase causes tri-enzyme inactivation. The cost of tri-enzymes is also very high. These problems prevent the utilization of this enzyme toolbox in practice. To address this, galactose oxidase, catalase, and horseradish peroxidase were co-immobilized into Cu(PO) nanoflowers in this study. The resulting co-immobilized tri-enzymes possessed better tolerance towards the oxidative damage caused by hydroxymethylfurfural at high concentrations, as compared to free tri-enzymes. Moreover, the 2,5-diformylfuran yield of co-immobilized tri-enzymes (95.7 ± 2.7%) was 1.06 times higher than that of separately immobilized enzymes (90.4 ± 1.9%). This result could be attributed to the boosted protective effect provided by catalase to the activity of galactose oxidase, owing to the physical proximity between them on the same support. After 30 recycles, co-immobilized tri-enzymes still achieves 86% of the initial yield. Moreover, co-immobilized tri-enzymes show enhanced thermal stability compared with free tri-enzymes. This work paves the way for the production of 2,5-diformylfuran from hydroxymethylfurfural via co-immobilized tri-enzymes.
Topics: Catalase; Enzymes, Immobilized; Furaldehyde; Furans; Glucose Oxidase; Horseradish Peroxidase; Oxidation-Reduction
PubMed: 31658589
DOI: 10.3390/molecules24203648 -
Cancer Medicine Jun 2016Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has...
Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has been shown to activate the plant hormone indole-3-acetic acid (IAA) to a powerful anticancer agent in in vitro studies, but gene directed enzyme prodrug therapy (GDEPT) studies showed ambivalent results. Thus, HRP/IAA in antibody directed enzyme prodrug therapy (ADEPT) was investigated as an alternative. However, this approach has not been intensively studied, since the enzyme preparation from plant describes an undefined mixture of isoenzymes with a heterogenic glycosylation pattern incompatible with the human system. Here, we describe the recombinant production of the two HRP isoenzymes C1A and A2A in a Pichia pastoris benchmark strain and a glyco-engineered strain with a knockout of the α-1,6-mannosyltransferase (OCH1) responsible for hypermannosylation. We biochemically characterized the enzyme variants, tested them with IAA and applied them on cancer cells. In the absence of H2 O2 , HRP C1A turned out to be highly active with IAA, independent of its surface glycosylation. Subsequent in vitro cytotoxicity studies with human T24 bladder carcinoma and MDA-MB-231 breast carcinoma cells underlined the applicability of recombinant HRP C1A with reduced surface glycoslyation for targeted cancer treatment. Summarizing, this is the first study describing the successful use of recombinantly produced HRP for targeted cancer treatment. Our findings might pave the way for an increased use of the powerful isoenzyme HRP C1A in cancer research in the future.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Enzyme Activation; Horseradish Peroxidase; Humans; Indoleacetic Acids; Inhibitory Concentration 50; Isoenzymes; Prodrugs; Recombinant Proteins
PubMed: 26990592
DOI: 10.1002/cam4.668 -
Analytical Sciences : the International... Apr 2022Horseradish peroxidase (HRP) is an enzyme that is frequently employed in various assays because HRP catalyzes the oxidation reactions of chromogenic and fluorogenic...
Horseradish peroxidase (HRP) is an enzyme that is frequently employed in various assays because HRP catalyzes the oxidation reactions of chromogenic and fluorogenic compounds to produce chromophores and fluorophores, respectively. The results of this study show that N-benzoyl leucomethylene blue (BLMB) is an excellent substrate for enzyme assay using HRP. In the presence of hydrogen peroxide (HO), HRP catalyzed an oxidation reaction of BLMB that produced methylene blue with a deep blue color. Thus, absorption spectrophotometry and capillary electrophoresis-laser-induced fluorometry (CE-LIF) could be used to easily determine the produced methylene blue. Under the optimum conditions, absorption spectrophotometry showed a linear calibration curve that ranged from 25 to 500 µg mL. The reaction conditions were also applicable to CE-LIF, showing a linear range of from 25 to 500 µg mL with limits of detection and quantification at 2 and 6 µg mL, respectively.
Topics: Electrophoresis, Capillary; Fluorometry; Horseradish Peroxidase; Hydrogen Peroxide; Lasers; Methylene Blue; Oxidation-Reduction; Spectrophotometry
PubMed: 35286641
DOI: 10.1007/s44211-022-00078-7 -
Chemical Research in Toxicology Jun 2021The fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study...
The fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study acellular reactive oxygen species (ROS) production from nanoparticles (NPs). This study examined whether HRP adsorbs onto NPs of Mn, Ni, and Cu and if this surface process influences the extent of metal release and hence the ROS production measurements using the DCFH assay in phosphate buffered saline (PBS), saline, or Dulbecco's modified Eagle's medium (DMEM). Adsorption of HRP was evident onto all NPs and conditions, except for Mn NPs in PBS. The presence of HRP resulted in an increased release of copper from the Cu NPs in PBS and reduced levels of nickel from the Ni NPs in saline. Both metal ions in solution and the adsorption of HRP onto the NPs can change the activity of HRP and thus influence the ROS results. The effect of HRP on the NP reactivity was shown to be solution chemistry dependent. Most notable was the evident affinity/adsorption of phosphate toward the metal NPs, followed by a reduced adsorption of HRP, the concomitant reduction in released manganese from the Mn NPs, and increased levels of released metals from the Cu NPs in PBS. Minor effects were observed for the Ni NPs. The solution pH should be monitored since the release of metals can change the solution pH and the activity of HRP is known to be pH-dependent. It is furthermore essential that solution pH adjustments are made following the addition of NaOH during diacetyl removal of DCFH-DA. Even though not observed for the given exposure conditions of this study, released metal ions could possibly induce agglomeration or partial denaturation of HRP, which in turn could result in steric hindrance for HO to reach the active site of HRP. This study further emphasizes the influence of HRP on the background kinetics, its solution dependence, and effects on measured ROS signals. Different ways of correcting for the background are highlighted, as this can result in different interpretations of generated results. The results show that adsorption of HRP onto the metal NPs influenced the extent of metal release and may, depending on the investigated system, result in either under- or overestimated ROS signals if used together with the DCFH assay. HRP should hence be used with caution when measuring ROS in the presence of reactive metallic NPs.
Topics: Adsorption; Fluoresceins; Horseradish Peroxidase; Metal Nanoparticles; Metals, Heavy; Particle Size; Reactive Oxygen Species; Surface Properties
PubMed: 33856197
DOI: 10.1021/acs.chemrestox.0c00430