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International Journal of Molecular... Jan 2018Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from...
Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic codon-adapted full-length isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the gene were synthesized, and their expression in was evaluated using an -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.
Topics: Codon; Horseradish Peroxidase; Recombinant Proteins; Nicotiana
PubMed: 29301255
DOI: 10.3390/ijms19010115 -
Molecules (Basel, Switzerland) Aug 2023Black phosphorene quantum dots (BPQDs) were prepared by ultrasonic-assisted liquid-phase exfoliation and centrifugation with morphologies proved by TEM results....
Black phosphorene quantum dots (BPQDs) were prepared by ultrasonic-assisted liquid-phase exfoliation and centrifugation with morphologies proved by TEM results. Furthermore, an electrochemical enzyme sensor was prepared by co-modification of BPQDs with horseradish peroxidase (HRP) on the surface of a carbon ionic liquid electrode (CILE) for the first time. The direct electrochemical behavior of HRP was studied with a pair of well-shaped voltammetric peaks that appeared, indicating that the existence of BPQDs was beneficial to accelerate the electron transfer rate between HRP and the electrode surface. This was due to the excellent properties of BPQDs, such as small particle size, high interfacial reaction activity, fast conductivity, and good biocompatibility. The presence of BPQDs on the electrode surface provided a fast channel for direct electron transfer of HRP. Therefore, the constructed electrochemical HRP biosensor was firstly used to investigate the electrocatalytic behavior of trichloroacetic acid (TCA) and potassium bromate (KBrO), and the wide linear detection ranges of TCA and KBrO were 4.0-600.0 mmol/L and 2.0-57.0 mmol/L, respectively. The modified electrode was applied to the actual samples detection with satisfactory results.
Topics: Quantum Dots; Carbon; Centrifugation; Electrodes; Horseradish Peroxidase
PubMed: 37630403
DOI: 10.3390/molecules28166151 -
Mikrochimica Acta Mar 2022Fe-based metal-organic framework (MIL-101(Fe)) was synthesized through a simple solvothermal synthesis and then used to prepare the AuNPs-decorated MIL-101(Fe)...
Fe-based metal-organic framework (MIL-101(Fe)) was synthesized through a simple solvothermal synthesis and then used to prepare the AuNPs-decorated MIL-101(Fe) nanocomposite (APPPM(Fe)) by a multi-step layer-by-layer assembly process. Benefited from the porous structure of MIL-101(Fe) and the multilayer assemble process, the loading amount of AuNPs on APPPM(Fe) was enhanced and exhibited a fine biocompatible interface and high conductivity. Through the intense Au-S bond, high loading amount of horseradish peroxidase was immobilized on APPPM(Fe) and the native bioactivity of HRP was kept to realize its direct electrochemistry. From the electrochemical kinetics, the constructed biosensor displayed fast electron transfer and good electrocatalysis activity for the detection of nitric oxide (NO) with wide linear range from 0.033 to 5370 μM and a low detection limit of 0.01 μM (3 σ) as well as fine stability, reproducibility and specificity. According to results of real sample analysis, the proposed electrochemical biosensor offers fast and simple detection of NO in real serum. Therefore, the present strategy definitely provided a potential application prospect in NO clinic detection and disease therapy.
Topics: Biosensing Techniques; Electrodes; Gold; Horseradish Peroxidase; Metal Nanoparticles; Metal-Organic Frameworks; Nitric Oxide; Reproducibility of Results
PubMed: 35348908
DOI: 10.1007/s00604-022-05268-8 -
Tissue Engineering. Part C, Methods Jun 2020Horseradish peroxidase (HRP) has been investigated as a catalyst to crosslink tissue-engineered hydrogels because of its mild reaction conditions and ability to modulate...
Horseradish peroxidase (HRP) has been investigated as a catalyst to crosslink tissue-engineered hydrogels because of its mild reaction conditions and ability to modulate the mechanical properties of the matrix. Here, we report the results of the first study investigating the use of HRP to crosslink fibrin scaffolds. We examined the effect of varying HRP and hydrogen peroxide (HO) incorporation strategies on the resulting crosslink density and structural properties of fibrin in a microthread scaffold format. Primary (1°) and secondary (2°) scaffold modification techniques were evaluated to crosslink fibrin microthread scaffolds. A primary scaffold modification technique was defined as incorporating crosslinking agents into the microthread precursor solutions during extrusion. A secondary scaffold modification technique was defined as incubating the microthreads in a postprocessing crosslinker bath. Fibrin microthreads were enzymatically crosslinked through primary, secondary, or a combination of both approaches. All fibrin microthread scaffolds crosslinked with HRP and HO via primary and/or secondary methods exhibited an increase in dityrosine crosslink density compared with uncrosslinked control microthreads, demonstrated by scaffold fluorescence. Fourier transform infrared spectroscopy indicated the formation of isodityrosine bonds in 1° HRP crosslinked microthreads. Characterization of tensile mechanical properties revealed that all HRP crosslinked microthreads were significantly stronger than control microthreads. Primary (1°) HRP crosslinked microthreads also demonstrated significantly slower degradation than control microthreads, suggesting that incorporating HRP and HO during extrusion yields scaffolds with increased resistance to proteolytic degradation. Finally, cells seeded on HRP crosslinked microthreads retained a high degree of viability, demonstrating that HRP crosslinking yields biocompatible scaffolds that are suitable for tissue engineering. The goal of this work was to facilitate the logical design of enzymatically crosslinked fibrin microthreads with tunable structural properties, enabling their application for engineered tissue constructs with varied mechanical and structural properties.
Topics: Animals; Biocompatible Materials; Cross-Linking Reagents; Fibrin; Horseradish Peroxidase; Humans; Hydrogels; Hydrogen Peroxide; Materials Testing; Tensile Strength; Tissue Engineering; Tissue Scaffolds
PubMed: 32364015
DOI: 10.1089/ten.TEC.2020.0083 -
Biomolecules Nov 2022The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local...
The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm HO for 30 min. Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.
Topics: Gelatin; Nanofibers; Horseradish Peroxidase; Hydrogen Peroxide; Plasmids; DNA
PubMed: 36358988
DOI: 10.3390/biom12111638 -
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 -
Trends in Biotechnology Aug 2006Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful biotechnological tools. HRP is often termed the classical plant heme peroxidase and although... (Review)
Review
Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful biotechnological tools. HRP is often termed the classical plant heme peroxidase and although it has been studied for decades, our understanding has deepened since its cloning and subsequent expression, enabling numerous mutational and protein engineering studies. SBP, however, has been neglected until recently, despite offering a real alternative to HRP: SBP actually outperforms HRP in terms of stability and is now used in numerous biotechnological applications, including biosensors. Review of both is timely. This article summarizes and discusses the main insights into the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and outlines its use in a variety of applications. It also reviews the current knowledge and applications to date of SBP, particularly biosensors. The final paragraphs speculate on the future of plant heme-based peroxidases, with probable trends outlined and explored.
Topics: Armoracia; Biosensing Techniques; Enzyme Activation; Horseradish Peroxidase; Mutagenesis, Site-Directed; Peroxidases; Recombinant Proteins; Glycine max; Structure-Activity Relationship
PubMed: 16815578
DOI: 10.1016/j.tibtech.2006.06.007 -
Biosensors Oct 2022High-performance electrochemical biosensors for the rapid detection of aflatoxin B1 (AFB) are urgently required in the food industry. Herein, a multi-scaled...
High-performance electrochemical biosensors for the rapid detection of aflatoxin B1 (AFB) are urgently required in the food industry. Herein, a multi-scaled electrochemical biosensor was fabricated by assembling carboxylated polystyrene nanospheres, an aptamer and horseradish peroxidase into a free-standing carbon nanofiber/carbon felt support. The resulting electrochemical biosensor possessed an exceptional performance, owing to the unique structures as well as the synergistic effects of the components. The 3D porous carbon nanofiber/carbon felt support served as an ideal substrate, owing to the excellent conductivity and facile diffusion of the reactants. The integration of carboxylated polystyrene nanospheres with horseradish peroxidase was employed as a signal amplification probe to enhance the electrochemical responses via catalyzing the decomposition of hydrogen peroxide. With the aid of the aptamer, the prepared sensors could quantitatively detect AFB in wine and soy sauce samples via differential pulse voltammetry. The recovery rates of AFB in the samples were between 87.53% and 106.71%. The limit of detection of the biosensors was 0.016 pg mL. The electrochemical biosensors also had excellent sensitivity, reproducibility, specificity and stability. The synthetic strategy reported in this work could pave a new route to fabricate high-performance electrochemical biosensors for the detection of mycotoxins.
Topics: Aflatoxin B1; Electrochemical Techniques; Hydrogen Peroxide; Reproducibility of Results; Polystyrenes; Carbon Fiber; Biosensing Techniques; Horseradish Peroxidase; Limit of Detection; Aptamers, Nucleotide
PubMed: 36291034
DOI: 10.3390/bios12100897 -
Molecules (Basel, Switzerland) Jun 2022Naringenin is one of the flavonoids originating from citrus fruit. This polyphenol is mainly found in grapefruit, orange and lemon. The antioxidant and antimicrobial...
Naringenin is one of the flavonoids originating from citrus fruit. This polyphenol is mainly found in grapefruit, orange and lemon. The antioxidant and antimicrobial properties of flavonoids depend on their structure, including the polymeric form. The aim of this research was to achieve enzymatic polymerization of naringenin and to study the properties of poly(naringenin). The polymerization was performed by methods using two different enzymes, i.e., laccase and horseradish peroxidase (HRP). According to the literature data, naringenin had not been polymerized previously using the enzymatic polymerization method. Therefore, obtaining polymeric naringenin by reaction with enzymes is a scientific novelty. The research methodology included analysis of the structure of poly(naringenin) by NMR, GPC, FTIR and UV-Vis and its morphology by SEM, as well as analysis of its properties, i.e., thermal stability (DSC and TGA), antioxidant activity (ABTS, DPPH, FRAP and CUPRAC) and antimicrobial properties. Naringenin oligomers were obtained as a result of polymerization with two types of enzymes. The polymeric forms of naringenin were more resistant to thermo-oxidation; the final oxidation temperature T of naringenin catalyzed by laccase (poly(naringenin)-laccase) was 28.2 °C higher, and poly(naringenin)-HRP 23.6 °C higher than that of the basic flavonoid. Additionally, due to the higher molar mass and associated increase in OH groups in the structure, naringenin catalyzed by laccase (poly(naringenin)-laccase) showed better activity for scavenging ABTS radicals than naringenin catalyzed by HRP (poly(naringenin)-HRP) and naringenin. In addition, poly(naringenin)-laccase at a concentration of 5 mg/mL exhibited better microbial activity against than monomeric naringenin.
Topics: Antioxidants; Citrus; Escherichia coli; Flavonoids; Horseradish Peroxidase; Laccase; Oxidation-Reduction; Polymers
PubMed: 35744827
DOI: 10.3390/molecules27123702 -
The Journal of Histochemistry and... Jan 2023This commentary briefly reviews the background for the development of the horseradish peroxidase-diaminobenzidine tetrahydrochloride histochemical method originally...
This commentary briefly reviews the background for the development of the horseradish peroxidase-diaminobenzidine tetrahydrochloride histochemical method originally described by Graham and Karnovsky in their citation classic, reprinted in full in this issue of . Some of the method's subsequent applications, including its use as a macromolecular tracer for kidney glomerular permeability and use in immunoelectron microscopy and other immunoassays, are also discussed.
Topics: Horseradish Peroxidase; Histocytochemistry
PubMed: 36541711
DOI: 10.1369/00221554221146838