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Molecules (Basel, Switzerland) Dec 2022Metal-organic frames (MOFs) have recently been used to support redox enzymes for highly sensitive and selective chemical sensors for small biomolecules such as oxygen...
Metal-organic frames (MOFs) have recently been used to support redox enzymes for highly sensitive and selective chemical sensors for small biomolecules such as oxygen (O), hydrogen peroxide (HO), etc. However, most MOFs are insulative and their three-dimensional (3D) porous structures hinder the electron transfer pathway between the current collector and the redox enzyme molecules. In order to facilitate electron transfer, here we adopt two-dimensional (2D) metal-organic layers (MOLs) to support the HRP molecules in the detection of HO. The correlation between the current response and the HO concentration presents a linear range from 7.5 μM to 1500 μM with a detection limit of 0.87 μM (S/N = 3). The sensitivity, reproducibility, and stability of the enzyme sensor are promoted due to the facilitated electron transfer.
Topics: Horseradish Peroxidase; Hydrogen Peroxide; Biosensing Techniques; Reproducibility of Results; Enzymes, Immobilized
PubMed: 36500690
DOI: 10.3390/molecules27238599 -
Glycobiology Sep 2014When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation...
When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.
Topics: Amino Acid Motifs; Biotechnology; Glycosylation; Horseradish Peroxidase; Mutation; Pichia; Plant Proteins; Protein Engineering; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 24859724
DOI: 10.1093/glycob/cwu047 -
BMC Biotechnology Dec 2007Horseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised...
BACKGROUND
Horseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications.
RESULTS
We have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated Sepharosetrade mark was noted with increased lysine content; however, only marginal gains in solvent stability resulted from immobilisation on this latter matrix.
CONCLUSION
Directional, orientated, immobilisation of rHRP mutants onto an activated, modified polyethersulfone membrane has been achieved with excellent retention of catalytic activity; however, re-engineering of acceptable stability characteristics into the "immobilisation mutants" will determine their applicability in diagnosis and biosensor development.
Topics: Amino Acid Substitution; Arginine; Binding Sites; Catalysis; Enzyme Stability; Enzymes, Immobilized; Horseradish Peroxidase; Lysine; Mutagenesis, Site-Directed; Recombinant Proteins
PubMed: 18053254
DOI: 10.1186/1472-6750-7-86 -
International Journal of Molecular... Jun 2020Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only...
Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.
Topics: Biotechnology; Escherichia coli; Escherichia coli Proteins; Horseradish Peroxidase; Inclusion Bodies; Protein Engineering; Recombinant Proteins
PubMed: 32610584
DOI: 10.3390/ijms21134625 -
The Biochemical Journal Dec 1997Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of...
Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.
Topics: Binding Sites; Catalysis; Dimerization; Dioctyl Sulfosuccinic Acid; Horseradish Peroxidase; Micelles; Models, Molecular; Protein Conformation; Recombinant Proteins; Structure-Activity Relationship; Surface-Active Agents
PubMed: 9371726
DOI: 10.1042/bj3280643 -
International Journal of Molecular... Aug 2022Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform...
Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.
Topics: Animals; Caco-2 Cells; Disulfides; Gastrointestinal Tract; Horseradish Peroxidase; Humans; Mice; Nanoparticles
PubMed: 36077259
DOI: 10.3390/ijms23179856 -
Analytical Sciences : the International... Feb 2001Horseradish peroxidase was incorporated in a kieselguhr membrane. The electron-transfer process of the enzyme was examined by cyclic voltammetry. It was observed that...
Horseradish peroxidase was incorporated in a kieselguhr membrane. The electron-transfer process of the enzyme was examined by cyclic voltammetry. It was observed that the electron-transfer reactivity of horseradish peroxidase was greatly enhanced, and that direct electrochemistry was accordingly feasible. Using the merits of the direct electron-transfer reactivity of horseradish peroxidase and its specific enzymatic catalysis towards hydrogen peroxide, an unmediated hydrogen peroxide biosensor was constructed. The calibration plot of this hydrogen peroxide sensor was linear in the range of 2.0 x 10(-6) mol/L - 6.5 x 10(-4) mol/L. The relative standard deviation was 4.1% for 6 successive determinations at a concentration of 1.0 x 10(-4) mol/L. The detection limit was 1.0 x 10(-6) mol/L.
Topics: Biosensing Techniques; Catalysis; Diatomaceous Earth; Electrochemistry; Enzymes, Immobilized; Horseradish Peroxidase; Hydrogen Peroxide; Hydrogen-Ion Concentration; Membranes, Artificial; Oxidation-Reduction
PubMed: 11990539
DOI: 10.2116/analsci.17.273 -
International Journal of Molecular... Feb 2023Limited membrane permeability and biodegradation hamper the intracellular delivery of the free natural or recombinant enzymes necessary for compensatory therapy....
Limited membrane permeability and biodegradation hamper the intracellular delivery of the free natural or recombinant enzymes necessary for compensatory therapy. Nanoparticles (NP) provide relative protein stability and unspecific endocytosis-mediated cellular uptake. Our objective was the fabrication of NP from 7 biomedicine-relevant enzymes, including DNase I, RNase A, trypsin, chymotrypsin, catalase, horseradish peroxidase (HRP) and lipase, the analysis of their conformation stability and enzymatic activity as well as possible toxicity for eukaryotic cells. The enzymes were dissolved in fluoroalcohol and mixed with 40% ethanol as an anti-solvent with subsequent alcohol evaporation at high temperature and low pressure. The shapes and sizes of NP were determined by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Enzyme conformations in solutions and in NP were compared using circular dichroism (CD) spectroscopy. The activity of the enzymes was assayed with specific substrates. The cytotoxicity of the enzymatic NP (ENP) was studied by microscopic observations and by using an MTT test. Water-insoluble ENP of different shapes and sizes in a range 50-300 nm consisting of 7 enzymes remained stable for 1 year at +4 °C without any cross-linking. CD spectroscopy of the ENP permitted us to reveal changes in proportions of α-helixes, β-turns and random coils in comparison with fresh enzyme solutions in water. Despite the minor conformation changes of the proteins in the ENP, the enzymes retained their substrate-binding and catalytic properties. Among the studied bioactive ENP, only DNase NP were highly toxic for 3 cell lines with granulation in 1 day posttreatment, whereas other NP were less toxic (if any). Taken together, the enzymes in the stable ENP retained their catalytic activity and might be used for intracellular delivery.
Topics: Antioxidants; Endopeptidases; Horseradish Peroxidase; Lipase; Nanoparticles; Peptide Hydrolases; Biocatalysis; Substrate Specificity
PubMed: 36769367
DOI: 10.3390/ijms24033043 -
Archives of Biochemistry and Biophysics Apr 2017A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate...
A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-diketogulonate (DKG), preparations of which can non-enzymically generate HO and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate HO and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λ 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major HO-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pK ≈ 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula CHO, i.e. it is a reduction product of DKG (CHO). In conclusion, compound (1) is the major HO-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate → dehydroascorbic acid → DKG → (1). We hypothesise that (1) generates apoplastic HO (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence.
Topics: 2,3-Diketogulonic Acid; Horseradish Peroxidase; Hydrogen Peroxide
PubMed: 28315301
DOI: 10.1016/j.abb.2017.03.006 -
The Biochemical Journal Jun 1990Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The... (Comparative Study)
Comparative Study
Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.
Topics: Benzene Derivatives; Chrysosporium; Electron Spin Resonance Spectroscopy; Horseradish Peroxidase; Hydrogen-Ion Concentration; Kinetics; Laccase; Mitosporic Fungi; Oxidoreductases; Peroxidases; Spectrophotometry, Ultraviolet
PubMed: 2163614
DOI: 10.1042/bj2680475