Stability Of Horseradish Peroxidase Research Articles

A self-assembled 3D nanoflowers based nano-ELISA platform for the sensitive detection of pyridaben

Biomimetic methods are invariably employed to synthesize hybrid organic–inorganic multilevel structure nanoflowers with self-assembly processes in aqueous solutions, which is an ideal way to meet the challenges of immobilizing antibodies or enzymes in nanomaterial based enzyme-linked immunosorbent assay (nano-ELISA). In this study, we developed protein-inorganic hybrid 3D nanoflowers composed of bovine serum albumin (BSA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (IgG-HRP) and copper(Ⅱ) phosphate (BSA-(IgG-HRP)-Cu3(PO4)2) using a self-assembly biomimetic method. The preparation process avoided the use of any organic solvent and protein immobilization did not require covalent modifications. Additionally, the unique hierarchical structure enhances the thermal and storage stability of HRP. The BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower was then applied to a nano-ELISA platform for pyridaben detection, achieving a 50% inhibition concentration of 3.90 ng mL−1. The nano-ELISA achieved excellent accuracy for pyridaben detection. Such a novel BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower provide an excellent reagent for small molecule immunoassay.

Glycosylation increases active site rigidity leading to improved enzyme stability and turnover.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRP's thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long-range effects that mediate the 'native' enzyme's activity and stability through changes in inherent dynamics.

Development of self-cooperative nanochaperones with enhanced activity to facilitate protein refolding.

Regulating protein folding including assisting de novo folding, preventing misfolding and aggregation, and facilitating refolding of proteins are of significant importance for retaining protein's biological activities. Here, we report a mixed shell polymeric micelle (MSPM)-based self-cooperative nanochaperone (self-CO-nChap) with enhanced activity to facilitate protein refolding. This self-CO-nChap was fabricated by introducing Hsp40-mimetic artificial carriers into the traditional nanochaperone to cooperate with the Hsp70-mimetic confined hydrophobic microdomains. The artificial carrier facilitates transfer and immobilization of client proteins into confined hydrophobic microdomains, by which significantly improving self-CO-nChap's capability to inhibit unfolding and aggregation of client proteins, and finally facilitating refolding. Compared to traditional nanochaperones, the self-CO-nChap significantly enhances the thermal stability of horseradish peroxidase (HRP) epicyclically under harsher conditions. Moreover, the self-CO-nChap efficiently protects misfolding-prone proteins, such as immunoglobulin G (IgG) antibody from thermal denaturation, which is hardly achieved using traditional nanochaperones. In addition, a kinetic partitioning mechanism was devised to explain how self-CO-nChap facilitates refolding by regulating the cooperative effect of kinetics between the nanochaperone and client proteins. This work provides a novel strategy for the design of protein folding regulatory materials, including nanochaperones.

Horseradish peroxidase-catalyzed synthesis of high-quality polyaniline in Good’s buffer ionic liquid-buffered H2O/AOT/isooctane bicontinuous microemulsion

Here, a functionalized organic salt composed of [Nk,k,k,k]+ (k = 1–4) and [MES]- (2-(N-morpholino)ethanesulfonate), i.e., Good’s buffer ionic liquid with self-buffering and biocompatible properties, was chosen as electrolyte as well as buffer to construct a H2O/AOT/isooctane bicontinuous microemulsion for enzymatic synthesis of polyaniline (PANI). Studies on the effects of the functionalized organic salt on the phase behavior indicate that [Nk,k,k,k][MES] can tune the phase inversion temperature of AOT-stabilized oil-water microemulsion system through changing the cationic alkyl chain length, exhibiting the properties of both inorganic salt and co-alcohol. More interestingly, the stability of horseradish peroxidase (HRP) solubilized in the H2O(0.1 M [N1,1,1,1][MES], pH = 4.5)/AOT/isooctane bicontinuous microemulsion is much better than that formulated with mixed surfactants AOT/SB-12 and inorganic salt. Studies on the HRP-catalyzed oxidative polymerization of aniline in the present bicontinuous microemulsion indicates that at low surfactant content and high oil-water ratio, the present microemulsion resulted in a PANI with better conductivity than the counterpart reported elsewhere. This work is of guiding significance for constructing a less-component and enzyme-compatible bicontinuous microemulsion for template-assisted biopolymerization.

Immobilization of horseradish peroxidase on lysine-functionalized gum Arabic-coated Fe3O4 nanoparticles for cholesterol determination

Background Horseradish Peroxidase (HRP) is ranked as one of the most important industrial enzymes that is extensively used in industry. Cholesterol is routinely detected indirectly by cholesterol oxidase in the presence of O2, liberating H2O2 as a by-product. The H2O2 content is determined through the HRP activity in the presence of a redox dye, producing a red colored quinoneimine which can be measured quantitatively. Herein, we have designed a magnetic nanoparticle for reusing and easily separating HRP as the most expensive compartment for the low-cost cholesterol assay. Methods The gum Arabic coated magnetic nanoparticles were functionalized with L-lysine linker for maintaining protein flexibility on nanoparticle. Enzyme-loaded nanoparticles were characterized by TEM, FTIR, DLS, VSM and XRD analysis. Results The immobilization efficiency was ∼65% and the immobilized HRP retained 60% of its activity after 8 times reuse. The optimum pH and thermal stability shifted from 7.0 to 8.0 and 60 to 70 °C after immobilization, respectively. Storage stability of HRP was improved by 10%, at 4 °C for 60 days. Immobilized HRP showed more catalytic activity in presence of Fe2+, Ca2+ and Na+. The designed system has cholesterol detection linearity range from 0.2 to 5.0 mM and detection limit of 0.08 mM and acceptable correlation coefficient of 0.9973 and 0.9982 on sample serum using both chromogens. Conclusion The HRP-loaded magnetic nanoparticles are capable of being used as a cost-effective system for cholesterol determination in laboratory due to its reusability and stability benefits.

Anionic-Surfactant-Stabilized Hydrophobic Ionic-Liquid-Based Bicontinuous Microemulsion as a Medium for Enzymatic Oxidative Polymerization of Aniline.

The hydrophobic ionic liquid [C8mim][PF6] (1-octyl-3-methylimidazolium hexafluorophosphate)-based bicontinuous microemulsion stabilized by the anionic surfactant [C4mim][AOT] (1-butyl-3-methylimidazolium bis(2-ethylhexyl) sulfosuccinate) was first tried as a medium for horseradish peroxidase (HRP)-triggered oxidative polymerization of aniline. The effects of the mass ratio of [C8mim][PF6]-to-water (α), the mass fraction of [C4mim][AOT] in the total mixture (γ), and temperature (T) on the enzymatic polymerization were investigated using UV–vis–NIR absorption, electron spin resonance, and small-angle X-ray scattering spectroscopy techniques. The bicontinuous microemulsion is demonstrated to play a template role in the biosynthesis of polyaniline (PANI). The conductivity of the resulting PANI depends on the microemulsion microstructure and the microstructure- and T-dependent catalytic properties of the solubilized HRP. With the increase in α, the conductivity of the synthesized PANI decreases due to the increase in the template curvature (decrease of the microdomain size) and the decrease in the activity and stability of HRP. Compared with α, γ has little effect on the microdomain size of the template; so, the γ-dependent change in the conductivity of PANI is mainly caused by the changes of the microstructure-dependent activity and stability of HRP. Over the range of 20–35 °C, T has little effect on the microdomain size, but it greatly changes the activity and stability of HRP. With the increase in T, the activity of HRP increases steadily, but its stability decreases significantly, which should be one of the reasons why the conductivity of PANI decreases with increasing T. In conclusion, lower values of α, γ, and T are favorable for the biosynthesis of conductive PANI. The present study not only deepens the insight into the role of the template in the process of PANI synthesis, but also opens up a green new way for the biosynthesis of the conducting polymer.

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards β-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

Non-invasive detection of glucose in human urine using a color-generating copper NanoZyme.

Renal complications are long-term effect of diabetes mellitus where glucose is excreted in urine. Therefore, reliable glucose detection in urine is critical. While commercial urine strips offer a simple way to detect urine sugar, poor sensitivity and low reliability limit their use. A hybrid glucose oxidase (GOx)/horseradish peroxidase (HRP) assay remains the gold standard for pathological detection of glucose. A key restriction is poor stability of HRP and its suicidal inactivation by hydrogen peroxide, a key intermediate of the GOx-driven reaction. An alternative is to replace HRP with a robust inorganic enzyme-mimic or NanoZyme. While colloidal NanoZymes show promise in glucose sensing, they detect low concentrations of glucose, while urine has high (mM) glucose concentration. In this study, a free-standing copper NanoZyme is used for the colorimetric detection of glucose in human urine. The sensor could operate in a biologically relevant dynamic linear range of 0.5-15mM, while showing minimal sample matrix effect such that glucose could be detected in urine without significant sample processing or dilution. This ability could be attributed to the Cu NanoZyme that for the first time showed an ability to promote the oxidation of a TMB substrate to its double oxidation diimine product rather than the charge-transfer complex product commonly observed. Additionally, the sensor could operate at a single pH without the need to use different pH conditions as used during the gold standard assay. These outcomes outline the high robustness of the NanoZyme sensing system for direct detection of glucose in human urine. Graphical abstract.

Storage stability of proteins in a liquid-based formulation: Liquid vs. solid self-emulsifying drug delivery.

The objective of the present study was the development of self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins providing storage stability. Horseradish peroxidase (HRP) serving as model protein was ion paired with docusate and incorporated into three liquid and three solid SEDDS formulations. Storage stability of HRP was determined over three weeks by quantifying its enzymatic activity. Generally, HRP maintained 78% of its initial enzymatic activity after complexation and loading into SEDDS. Having been incorporated in liquid SEDDS the protein showed limited stability and precipitated within a few hours. In contrast, in all solid SEDDS comprising of hard fats such as Witepsol W45 and solid surfactants such as Gelucire 44/14 and 48/16 as solidifying agents HRP was successfully stabilized. No decrease in HRP activity could be observed over the entire observation period. Solid SEDDS based on high-melting components can provide storage stability of incorporated proteins, whereas liquid SEDDS cannot.

Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase

In this report, activity and stability of horseradish peroxidase (HRP) entrapped in polyacrylamide gel in the presence of proline (HEP) are compared with that of enzyme entrapped in absence of proline (HE). Within polyacrylamide (8%) beads, 80% entrapment yield for peroxidase was observed in the presence as well as absence of proline. The HEP (1.5M proline) showed 170% higher activity compared to HE. HEP also showed significant increase in K M, V max and K cat. At 8th cycle of use, HEP retained 40% of its activity, whereas HE retained only 10% of activity. In addition, in comparison with HE, HEP showed increased storage stability and thermo-stability. HEP showed higher activity compared to HE over an extensive range of pH (4-8), temperature (30-80°C) and inhibitors such as NaN3, Cd2+ and Pb2+. Our results suggest that peroxidase entrapment in polyacrylamide gel in the presence of proline can be a useful approach for increasing activity and stability of horseradish peroxidase.

Horseradish peroxidase-catalyzed oxidative polymerization of aniline in bicontinuous microemulsion stabilized by AOT/SDS

A bicontinuous microemulsion system consisting of sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), an aqueous solution (a 0.1 M disodium hydrogenphosphate/citric acid buffer solution of pH = 4.3), and decane was investigated in detail for applications as reaction medium for the horseradish peroxidase (HRP)/hydrogen peroxide-mediated synthesis of the conductive form of polyaniline (PANI). To formulate the optimal single-phase microemulsion with the phase inversion temperature being close to room temperature, the T-γ fishlike phase diagram of the AOT/SDS-buffer-decane pseudoternary system was determined. The effects of the surfactant concentration (γ), the oil-to-water ratio (α) and the temperature (T) on the conductivity of the resulting PANI and the underlying mechanism were investigated using UV–vis-NIR, ESR and small-angle X-ray scattering (SAXS) techniques. Results show that the bicontinuous microemulsion plays a template role in the PANI biosynthesis, and the PANI conductivity also depends on the activity and stability of the solubilized HRP, which are determined by the microdomain size of the microemulsions and the reaction temperature. As far as γ is concerned, it is found that as γ increases, the microdomain size of the bicontinuous microemulsion as well as the activity of the solubilized HRP decreases significantly, leading to a great decrease of the PANI conductivity. Compared with γ, the change of α has small effect on the PANI conductivity. As the α-dependent microdomain size changes little with increasing α, the small change of the PANI conductivity should be ascribed mainly to the HRP activity. For T, over the range of 20 °C–35 °C, it has a slight effect on the microdomain size of the bicontinuous microemulsion, but it has a marked effect on the enzymatic properties of HRP. As the temperature increases, the activity of HRP increases, but its thermal stability decreases greatly. Therefore, the thermal stability of HRP is the major factor causing that the PANI conductivity decreases significantly with increasing T. The present work deepens the understanding of the template effect of anionic surfactant aggregates in the biosynthesis of conducting PANI. To the best of our knowledge, this work is the first study of the use of bicontinuous microemulsions as reaction media for the enzymatic synthesis of a conducting polymer.

Single-Atom Nanozymes Linked Immunosorbent Assay for Sensitive Detection of Aβ 1-40: A Biomarker of Alzheimer's Disease.

Single-atom nanozymes (SANs) possess unique features of maximum atomic utilization and present highly assembled enzyme-like structure and remarkable enzyme-like activity. By introducing SANs into immunoassay, limitations of ELISA such as low stability of horseradish peroxidase (HRP) can be well addressed, thereby improving the performance of the immunoassays. In this work, we have developed novel Fe-N-C single-atom nanozymes (Fe-Nx SANs) derived from Fe-doped polypyrrole (PPy) nanotube and substituted the enzymes in ELISA kit for enhancing the detection sensitivity of amyloid beta 1-40. Results indicate that the Fe-Nx SANs contain high density of single-atom active sites and comparable enzyme-like properties as HRP, owing to the maximized utilization of Fe atoms and their abundant active sites, which could mimic natural metalloproteases structures. Further designed SAN-linked immunosorbent assay (SAN-LISA) demonstrates the ultralow limit of detection (LOD) of 0.88 pg/mL, much more sensitive than that of commercial ELISA (9.98 pg/mL). The results confirm that the Fe-Nx SANs can serve as a satisfactory replacement of enzyme labels, which show great potential as an ultrasensitive colorimetric immunoassay.

The influence of rotating magnetic field on bio-catalytic dye degradation using the horseradish peroxidase

The aim of this study was to analyze the effects of a rotating magnetic field (RMF) on the activity and stability of horseradish peroxidase (HRP) and the catalyzed enzymatic process. It was found that 8 h of exposure to RMF does not influence the operational stability of HRP. At the same time, it was observed that the RMF improved HRP activity in a wider range of pH: 49% and 68% at pH 6.5 and pH 7.0, respectively. The efficiency of the catalyzed enzymatic process under exposure to RMF was analyzed by two-dimensional correlation analysis (2DCorr) on the example of the Remazol Brilliant Blue R (RBBR) decolorization process. The obtained results indicated the influence of RMF on the intensification of this process. Our studies showed an alternative method of enzymatic process intensification, which is significant in science and has practical value for applications.

Inhibition kinetic and thermal inactivation of horseradish peroxidase in the presence of Zn2+ ion

Peroxidase as a heme-containing metalloprotein, is an important enzyme for food industries. Hence, the current study is aimed to evaluate the inhibitory properties of zinc ion in terms of activity and thermal stability of horseradish peroxidase (HRP) in the range of 40-70°C for 1-45min. The results showed that zinc ion potently inhibited HRP activity by noncompetitive mechanism. The thermal inactivation of enzyme followed first-order reaction kinetics in the presence of ZnCl2 up to 300μM. However, higher concentrations of Zn2+ -induced irregularities in HRP thermo inactivation pattern from first-order kinetics. According to the kinetic parameters, K and Ea , the rate of reactivation of HRP may be dependent on the amount of zinc ion, inactivation time, and heating duration. PRACTICAL APPLICATIONS: Thermal inactivation kinetics of HRP was investigated in the presence of Zn2+ . The results showed that thermal inactivation of HRP followed first-order kinetics for up to 300μM of Zn2+ . HRP inactivation was consistent with biphasic models for higher concentrations of Zn2+ at 60°C. The transition free energy of inactivation confirmed that zinc ion increased the enzyme unfolding transition states stability as denatured structure of HRP is more favorable than native one in this condition. Moreover, reactivation potential of enzyme decreased by increasing the zinc ion concentration.

Paper Microzone Plates as Analytical Tools for Studying Enzyme Stability: A Case Study on the Stabilization of Horseradish Peroxidase Using Trehalose and SU-8 Epoxy Novolac Resin.

Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents.

Catalytic activity and thermal stability of horseradish peroxidase encapsulated in self-assembled organic nanotubes.

Horseradish peroxidase (HRP) was encapsulated in self-assembled lithocholic acid (LCA) based organic nanotubes and its catalytic activity before and after thermal treatment was measured for comparison with free HRP. The apparent kcat (kcat/Km) for nanotube encapsulated HRP remained almost the same before and after thermal treatment, reporting an average value of 3.7 ± 0.4 μM(-1) s(-1). The apparent kcat value for free HRP decreased from 14.8 ± 1.3 μM(-1) s(-1) for samples stored at 4 °C to 2.4 ± 0.1 μM(-1) s(-1) after thermal treatment for 8 h at 55 °C. The Michaelis-Menten constants, Km, determined for encapsulated HRP and free HRP were relatively unperturbed by storage conditions at 4 °C or thermally treated at 55 °C for varying time periods from 2-8 h, with encapsulated HRP having a slightly higher Km than free HRP (13.4 ± 0.9 μM versus 11.7 ± 0.4 μM). The amount of HRP encapsulated in LCA nanotubes increased dramatically when the mixture of HRP and LCA nanotubes was brought to an elevated temperature. Within 4 h of thermal treatment at 55 °C, the amount of HRP encapsulated by the LCA nanotubes was more than 4 times the amount of HRP encapsulated when equilibrated at 4 °C for 7 days. Molecular dynamics (MD) simulations show that the higher degree of exposure of hydrophobic residues in HRP at elevated temperatures enhances the hydrophobic interaction between HRP and the nanotube wall, resulting in the increased amount of HRP surface adsorption and, hence, the overall amount of encapsulation inside the nanotubes.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

2,4-Dichlorophenol removal by purified horseradish peroxidase enzyme and crude extract from horseradish immobilized to nano spray dried ethyl cellulose particles

Horseradish peroxidase (HRP) is a promising catalyst in the enzymatic process of phenolic compounds removal from wastewaters. Enzyme immobilization provides important benefits in a biotechnological process. Fine particles with a high surface-to-volume ratio composed of ethyl cellulose (EC) polymer were generated by Nano Spray Dryer B-90 as supports for HRP. Carrier particles were prepared by spray caps with hole size of 7μm, 5.5μm and 4μm. Purified HRP and crude extract from horseradish were covalently bound to the carrier using a carbodiimide cross-linker. The attached HRP content, the effect of pH on the activity and the storage stability were investigated. 2,4-dichlorophenol, an extremely persistent chlorinated phenol was removed by the immobilized enzyme, and the effect of main process parameters such as H2O2 and 2,4-dichlorophenol substrate concentrations were studied. After immobilization both the purified HRP and the horseradish extract performed better in the pH range of 4–10 and could preserve the activity substantially longer than the free enzyme. The immobilized enzyme was found to be outstandingly efficient (in optimal case close to 100%) in the elimination of 2,4-dichlorophenol, which was also the consequence of the high adsorbing capacity of the fine particles. The reuse study proved the operational stability of HRP attached to EC even after 10 consecutive cycles.

Reduced graphene oxide enhances horseradish peroxidase stability by serving as radical scavenger and redox mediator

Graphene-based nanomaterials have been widely studied as high-performance matrices for enzyme immobilization and in the development of biosensors. Surface O-functionalities of graphene induce changes in chemical reactivity and electronic conductivity of nanomaterials and may interfere with enzymatic processes; however, the mechanisms are not fully understood. We compare the effects of three commercially available graphene-based nanomaterials, namely a graphene, a graphene oxide (GO), and a reduced graphene oxide (RGO), on the activity/stability of horseradish peroxidase. Both graphene and GO significantly reduced enzyme stability by altering enzyme conformation, which was evidenced by circular dichroism spectroscopy. However, RGO improved enzyme stability up to 7-fold. This increased stability was attributed to the capability of RGO to quench superoxide radicals, which were primarily responsible for the enzyme deactivation. The basal plane of RGO, mainly through quinone moieties, may act as a redox mediator to facilitate enzymatic turnover. Overall, the radical scavenging plus redox mediating capabilities of RGO suggest the potential for graphene-based nanomaterials to enhance enzyme engineering and enzyme-based sensors.

Effect of PEGylation on the activity and stability of horseradish peroxidase and l-N-carbamoylase in aqueous phases

Effect of pegylation on the activities and stabilities of horseradish peroxidase (HRP) and n-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) was investigated. Pegylation was favored under alkaline conditions with high PEG/enzyme molar ratios. The lysine residues in l-N-carbamoylase were found to be more readily accessible for pegylation than those in HRP, leading to the formation of multi-pegylated enzymes. Pegylation and addition of mPEG as an additive improved HRP activity by 64% and 42%, respectively, via the formation of protective layers against the free radical-induced inactivation. The activity of pegylated l-N-carbamoylase, however, declined due to the additional mass transfer resistance introduced by the PEG layers. The extent of reduction in l-N-carbamoylase activity increased with the level of pegylation. In terms of thermostability, both the addition of mPEG as an additive and pegylation with PEG5000 led to a decline in thermostability. Nevertheless, pegylation of HRP with PEG2000 resulted in a PEG-HRP conjugate exhibiting a thermostability two-fold that of the control at 70°C, probably due to the capability of the relatively hydrophilic PEG domains on the conjugate in avoiding enzyme aggregation. After a 4-day incubation at room temperature, pegylated HRP retained significantly higher activity, 89%, than the control, 12%.