Encapsulation Of Horseradish Peroxidase Research Articles

Encapsulation of HRP-Immobilized Silica Particles into Hollow-Type Spherical Bacterial Cellulose Gel: A Novel Approach for Enzyme Reactions within Cellulose Gel Capsules

We revealed that the encapsulation of enzyme-immobilized silica particles in hollow-type spherical bacterial cellulose (HSBC) gels enables the use of the inside of HSBC gels as a reaction field. The encapsulation of horseradish peroxidase (HRP)-immobilized silica particles (Si-HRPs, particle size: 40–50 μm) within HSBC gels was performed by using a BC gelatinous membrane produced at the interface between Komagataeibacter xylinus suspension attached onto an alginate gel containing Si-HRPs and silicone oil. After the biosynthesis of the BC gelatinous membrane, formed from cellulose nanofiber networks, the alginate gel was removed via immersion in a phosphate-buffered solution. Si-HRP encapsulated HSBC gels were reproducibly produced using our method with a yield of over 90%. The pore size of the network structure of the BC gelatinous membrane was less than 1 μm, which is significantly smaller than the encapsulated Si-HRPs. Consequently, the encapsulated Si-HRPs could neither pass through the BC gelatinous membrane nor leak from the interior cavity of the HSBC gel. The activity of the encapsulated HRPs was detected using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system, demonstrating that this method can encapsulate the enzyme without inactivation. Since HSBC gels are composed of a network structure of biocompatible cellulose nanofibers, immune cells cannot enter the hollow interior, thus, the enzyme-immobilized particles encapsulated inside the HSBC gel are protected from immune-cell attacks. The encapsulation technique demonstrated in this study is expected to facilitate the delivery of enzymes and catalysts that are not originally present in the in vivo environment.

Enzyme activity assay for horseradish peroxidase encapsulated in peptide nanotubes

Encapsulation of horseradish peroxidase (HRP) inside a peptide nanotube (PNT) was demonstrated and its activity was measured. Enzyme assay verified that 0.16μg of the enzymes were encapsulated in 1mg of PNTs. The encapsulation was also verified with TEM, UV–vis spectroscopy, and FTIR. The activity of the encapsulated HRP was examined for thermal stability, long-term storage stability, and resistance to a denaturant. They showed good storage stability, retaining its activity up to 90%, while the free HRP lost 50% of its activity over the course of 18days. At 55°C, the encapsulated HRP activity remained 20% higher than that of the free HRP. With the denaturant, guanidinium hydrochloride (GdmHCl), the encapsulated HRP activity was maintained around 10% higher than the free HRP. This result proves that the encapsulation of HRP inside the PNT may be an effective way to keep the enzyme activity stable in various environments.

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions

Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (−20°C, 4°C, 25°C or 45°C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45°C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzyme's initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25°C or 45°C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.

Gold nanoparticles/horseradish peroxidase encapsulated polyelectrolyte nanocapsule for signal amplification in Listeria monocytogenes detection

Bioconjugate nanocapsules were fabricated by using polystyrene sulfonate (PSS) to encapsulate gold nanoparticles (AuNPs) bearing adsorbed horseradish peroxidase (HRP). The average size of nanocapsule was in a range 150–400nm. The efficiency of the capsules to enhance signals in an immunoassay was demonstrated by using an enzyme linked immunosorbent assay (ELISA) to detect the food-borne pathogen –Listeria monocytogenes. The antibody adsorbed onto the PSS shell of the nanocapsules provided the recognition molecule. For a given quantity of antibody, the bioconjugate nanocapsules showed 30 times greater sensitivity and a shorter assay time (5min) when compared to conventional ELISA using an HRP labelled antibody. This proof-of-concept encapsulation of HRP through PSS nanocapsules may pave the way for alternative signal enhancement strategies where sensitivity is a priority.

In situ enzymatic aided formation of xylan hydrogels and encapsulation of horse radish peroxidase for slow release

Xylans have special gelling and film forming properties for production of hydrogels that can be used as encapsulation matrices for slow release of bioactive agents. Hydrogels with particle sizes ranging from 18nm to >10μm and zeta potential of up to −19mV were formed by selective removal of arabinose side chains from oat spelt xylan by recombinant α-l-arabinofuranosidase (AbfB). The xylan concentration significantly (P<0.05) influenced both the particle size and zeta potential of the oat spelt xylan hydrogels, and the later was also influenced significantly (P<0.05) by the hydrolysis time. The horse radish peroxidase was encapsulated in situ, both during and after formation of the xylan hydrogels and was released in an active form for a period of 180min. Thus, the recombinant AbfB presents a biological tool for modification of xylan into biodegradable encapsulation matrices for slow or targeted release of bioactive agents.

An Amperometric Sensor for Nanomolar Detection of Hydrogen Peroxide Based on Encapsulation of Horseradish Peroxidase in Thymol Blue-Ormosil Composite

An Amperometric Sensor for Nanomolar Detection of Hydrogen Peroxide Based on Encapsulation of Horseradish Peroxidase in Thymol Blue-Ormosil Composite

Fluorescence Decrease of Conjugated Polymers by the Catalytic Activity of Horseradish Peroxidase and Its Application in Phenolic Compounds Detection

We report the fluorescence decrease of the water-soluble π-π-conjugated polymer poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene, MPS-PPV) by the catalytic activity of horseradish peroxidase in the presence of H(2)O(2). MPS-PPV acts as a donor substrate in the catalytic cycle of horseradish peroxidase where the electron-deficient enzymatic intermediates compounds I and II can subtract electrons from the polymer leading to its fluorescence decrease. The addition of phenolic drug acetaminophen to the former solution favors the decrease of the polymer fluorescence, which indicates the peroxidase-catalyzed co-oxidation of MPS-PPV and acetaminophen. The encapsulation of horseradish peroxidase within polyacrylamide microgels allows the isolation of intermediates compound I and compound II from the polymer, leading to a fluorescence decrease that is only due to the product of biocatalytic acetaminophen oxidation. This system could be used to develop a new device for phenolic compounds detection.

Encapsulation of horseradish peroxidase into hydrogel, and its bioelectrochemistry

A biohybrid hydrogel is fabricated by integrating horseradish peroxidase (HRP) with polyhydroxyl cellulose (PHC), which is prepared by mixing of poly(vinyl alcohol) and carboxymethyl hydroxyethyl cellulose. PHC provides a biocompatible microenvironment for HRP, and the UV-vis spectrum indicates that the confromation of HRP-loaded biohydrogel is well retained relative to free HRP. Based on the direct electrochemistry and electrocatalytic ability of HRP, a third-generation H2O2 biosensor is developed. Under the optimized conditions, the H2O2 biosensor shows a linear response over the range from 1.0 μM to 1.0 mM with a detection limit (S/N = 3) of 0.5 μM.

High operational stability in peroxidase-catalyzed non-aqueous sulfoxidations by encapsulation within sol–gel glasses

The peroxidase activity of immobilized horseradish peroxidase (HRP) has been employed for the asymmetric oxidation of thioanisole in acetonitrile with H 2O 2 (30%). Encapsulation of HRP by the sol–gel method considerably enhanced its operational stability by protecting the peroxidase activity under harsh conditions. The total rates of the encapsulated HRP increased up to six-fold (TTN=4.22×10 3) the rates observed with its homogeneous counterpart. The sulfoxide selectivity and the enantiomeric excess also increased greatly upon encapsulation. Coupling glucose oxidase reaction to the encapsulated peroxidase allowed high enantiomeric excesses (up to 56%) and sulfoxide as sole product by elimination of side non-enantioselective and overoxidation reactions. The heterogeneous catalyst can be recycled by simple filtration in successive runs.

Oxidation of ABTS by hydrogen peroxide catalyzed by horseradish peroxidase encapsulated into sol–gel glass.: Effects of glass matrix on reactivity

Encapsulation of horseradish peroxidase (HRP) by the sol–gel method into silica (SiO 2) or alkylated silica (RSiO 2, in which R is Me or Pr) yields biocatalytic glasses designated HRP@SiO 2 and HRP@RSiO 2. These new enzyme composite materials catalyze one-electron oxidation by H 2O 2 of the dye 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) into the radical cation ABTS +. In the presence of excess H 2O 2, ABTS + is converted into a mixture of at least five compounds. This undesirable side-reaction can be suppressed by using stoichiometric amount of H 2O 2. The effects of ABTS concentration and ionic strength of the buffer on the ABTS + yield and the apparent rate constant were investigated. The catalyst HRP@MeSiO 2 achieves higher ABTS + yields (70–85%) than HRP@SiO 2 and HRP@PrSiO 2 (30–40%) do. The apparent rate constants for HRP@MeSiO 2 are 7–10 times higher than those for HRP@SiO 2 and HRP@PrSiO 2. When the ionic strength of the buffer is raised, the rate constants increase for HRP@SiO 2 and HRP@PrSiO 2, and do not change for HRP@MeSiO 2. The diminished electrostatic interaction between the negatively-charged substrate, ABTS, and the surface of methylated silica is the main cause for the increased catalytic efficiency. These results are important for understanding of encapsulated and otherwise immobilized enzymes. The possible electrostatic effects should be taken into consideration in the choice of the matrix materials used in the design of biosensors, supported catalysts, and other composite materials.