Immobilization Of Peroxidase Research Articles

Experimental and computational evaluation of area selectively immobilized horseradish peroxidase in a microfluidic device

A microreactor with a square shaped reactor chamber was developed with the aim to correlate enzyme positioning with biocatalytic activity. The enzyme position as an important parameter to improve the contribution of the individual enzymes towards the overall reactor efficacy was therefore evaluated experimentally and by computational fluid dynamics (CFD) simulations. Ultimately, such a correlation would lead to faster development through computational pre-screening and optimized experimental design.In this proof-of-concept study, microreactors were prepared in a 2-step curing process of an off-stoichiometric thiol-ene-epoxy (OSTE+) mixture employing both a thiol-ene (TEC) and a thiol-epoxy curing reaction. Subsequent surface functionalization of the remaining thiol groups on the reactor surface through stenciled photoinitiated TEC enabled the preparation of specific surface patterns in the reactor. Patterns were visualized using an allyl-functional disperse red dye, illustrating the successful preparation of a fully reacted surface, a half covered surface and 2 checkerboard patterns. Similarly, allyl glycidyl ether was exploited to functionalize the microreactor surface with epoxide groups, which were used for covalent immobilization of horseradish peroxidase (HRP) in the same patterns. Biocatalytic activity measurements confirmed a clear dependency of the overall reactor performance depending on the spatial distribution of the immobilized enzymes, where specifically the two checkerboard motifs were identified as being particularly effective compared to enzymes covering homogeneously the entire reactor surface. The performance of the same configurations was additionally determined by 3-dimensional CFD simulations. The computational model predicted the same tendencies for the overall reactor performance as obtained from experimental determination. This good agreement between the obtained experimental and computational results confirmed the high potential of CFD models for predicting and optimizing the biocatalytic performance of such a reactor.

Nitrilotriacetic Amine-Functionalized Polymeric Core-Shell Nanoparticles as Enzyme Immobilization Supports.

Nitrilotriacetic amine (NTA)-functionalized nanoparticles obtained by aqueous polymerization-induced self-assembly (PISA) are introduced as immobilization supports for polyhistidine-functionalized (His-tagged) enzymes. A novel initiator for nitroxide-mediated polymerization based on the nitroxide SG1 and carrying a protected NTA moiety was first synthesized. Size-exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI-MS) proved the ability of this initiator to produce well-defined end-functional vinyl polymers. Subsequently, oligo(ethylene glycol) methacrylate-based macroinitiators were synthesized and chain-extended to form amphiphilic block copolymer nanoparticles, either by nanoprecipitation or by PISA. The latter method yielded spherical nanoparticles with a higher definition, as demonstrated by dynamic light scattering (DLS). Deprotection of the NTA moiety and complexation with nickel ions were assessed by DLS and inductively coupled plasma optical emission spectroscopy/mass spectrometry (ICP-OES/MS). Finally, immobilization of His-tagged horseradish peroxidase and ester hydrolase were successfully carried out, leading to catalytically active nanobiocatalysts, as shown by UV-vis measurements.

Immobilization of horseradish peroxidase on electrospun poly(vinyl alcohol)–polyacrylamide blend nanofiber membrane and its use in the conversion of phenol

Electrospun nanofibers, with their porous structures, high surface-to-volume ratio, and good mechanical properties, are used as a support material for enzyme immobilization. In this study, the poly(vinyl alcohol) and polyacrylamide bicomponent (PVA–PAAm) nanofibers were fabricated via the electrospinning method. Synthesized PAAm was characterized with size exclusion chromatography (SEC). Nanofibers were characterized by fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscope (SEM). DSC and TGA analyses showed that the nanofibers were more durable than PVA and PAAm polymers. SEM images demonstrated that all nanofibers possessed uniform and smooth structures (average diameter about 300 nm). FTIR results have shown that PAAm successfully participates in nanofiber structure. The produced nanofibers were used as support material for covalent immobilization of horseradish peroxidase (HRP). The optimum temperature for free HRP was 45 °C, whereas it was 50 °C for the immobilized enzyme. The immobilized HRP showed better storage and thermal stability than free HRP. The kinetic parameters (K m and V max) were found to be 2.42 mM and 0.027 U for the immobilized HRP and 1.86 mM and 0.079 U for the free HRP, respectively. The immobilized enzyme could be used effectively for 25 cycles with 54% retention of the activity. The immobilized HRP was also used for the conversion of phenol. Phenol removal was found to be about 29.68% at 180 min in real wastewater. The novel PVA–PAAm nanofibrous material was successfully used as a support material for covalent immobilization of HRP. Immobilized enzymes such as oxido-reductases onto the PVA–PAAm bicomponent nanofiber could be recommended in the treatment of organic pollutants in industrial effluents.

A colorimetric and electrochemical immunosensor for point-of-care detection of enterovirus 71

Point-of-care detection of human enterovirus 71 (EV71), the major pathogen that causes hand, foot, and mouth disease (HFMD) among children, is urgently needed for early diagnosis and control of related epidemics. A colorimetric and electrochemical immunosensor for point-of-care detection of EV71 has been developed based on dual-labeled magnetic nanobeads amplification. The dual-labeled magnetic nanobeads (DL-MBs) are fabricated by simultaneous immobilization of EV71 monoclonal antibody (mAb) and horseradish peroxidase (HRP) on magnetic nanobeads. By capturing EV71 virions in 20μL sample on mAb modified AuNPs-coated ITO electrode and subsequently binding with DL-MBs, with the addition of TMB and H2O2, colorimetric signals corresponding to EV71 with a concentration of 1.0ngmL−1 can be directly read out by naked eyes, making it possible towards point-of-care detection of the virus. Furthermore, on the reduction of oxidized TMB on the electrode, electrochemical signal can be detected in the same detection cell without solution transfer, with a detection limit of 0.01ngmL−1. Validated with clinical samples, the colorimetric and electrochemical immunosensor shows a complete consistency with reverse transcription-polymerase chain reaction (RT-PCR) results. So far as we know, this is the first report on EV71 detection using electrochemical method. The merits of this assay, including high sensitivity, ability for colorimetric detection and easy to operation, ensure a promising future in point-of-care diagnostics of virus related diseases.

A label-free electrochemical biosensor for methyltransferase activity detection and inhibitor screening based on graphene quantum dot and enzyme-catalyzed reaction

A label-free electrochemical biosensor for methyltransferase (M.SssI MTase) activity detection and inhibitor screening was developed based on the amplification of graphene quantum dot (GQD) and enzyme-catalyzed reaction. In the construction of the electrochemical biosensor, auxiliary DNA hybridized with capture DNA to form the double-stranded DNA structure which contained a specific recognition sequence (5′-CCGG-3′) for both M.SssI MTase and restriction endonuclease HpaII. The presence of M.SssI MTase caused the methylation of CpG site in the ds-DNA, which could not be digested by HpaII, causing GQD bound to the undigested ds-DNA and acted as new platform for the immobilization of horseradish peroxidase (HRP) through noncovalant assembly. The modified HRP catalyzed the hydrogen peroxide-mediated oxidation of 3, 3′, 5, 5′-tetramethylbenzidine, resulting an electrochemical signal output. The proposed biosensor realized sensitivity detection of M.SssI MTase activity in the range of 1–40UmL−1 with a detection limit of 0.3UmL−1, and this dual-amplified biosensor had been successfully applied in M.SssI MTase activity inhibitor screening of procaine and epicatechin. Furthermore, this proposed strategy can also be extended for other methyltransferase detection, which had a promising application in clinical diagnosis and drug development.

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5 h at 85 °C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75 °C, whereas that of the free enzyme was 55 °C. After two months at 4 °C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180 min and, after five cycles of use, the activity loss was only 12.8%.

A spick-and-span approach to the immobilization of horseradish peroxidase on Au nanospheres incorporated with a methionine/graphene biomatrix for the determination of endocrine disruptor bisphenol A

In the present study, we employ a straightforward, benign strategy to prepare thiol-functionalized reduced graphene oxide (S-RGO) using methionine as the sulphur source and reducing agent. The immobilization of horseradish peroxidase (HRP) over the AuNPs/S-RGO was developed by incorporating AuNPs on the S-RGO surface. The fabricated HRP/AuNPs/S-RGO electrode exhibits a remarkable decrease in the overpotential and a significantly increased oxidation peak current of bisphenol A (BPA) compared with the bare glassy carbon electrode (GCE) and AuNPs/S-RGO electrode. The biosensor shows an excellent amperometric analytical performance with a low detection limit of 2.6×10−12M and a linear range from 2.0×10−11 to 1.18×10−9M, with the response time <2s for BPA. From the results, the apparent Michaelis-Menten constant was calculated as 8.14nM. The HRP/AuNPs/S-RGO biosensor exhibited faster response, adequate storage stability, inexpensive, simple fabrication with disposability, satisfactory reproducibility and repeatability, and outstanding selectivity. Finally, the constructed biosensor was utilized successfully for detecting BPA in tomato juice and milk samples with acceptable results.

Immobilization of Euphorbia tirucalli peroxidase onto chitosan-cobalt oxide magnetic nanoparticles and optimization using response surface methodology

Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50°C and 55°C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2mg/286mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability.

Polyvinylamine-coated polyester fibers as a carrier matrix for the immobilization of peroxidases.

Biocatalytic transformations that employ immobilized enzymes become increasingly important for industrial applications. Synthetic or natural textile fiber materials such as polyester, polyamide or viscose are support materials that are comparatively inexpensive. Contrary to traditional support materials, their flexibility enables their use in reactors of any geometry and a fast and residue-free removal from batch reactors. In this study a permanently immobilized peroxidase (Baylase®) has been investigated on polyester felt as a solid support as a new heterogeneous catalyst system. The polyester felt was functionalized by coating with polyvinylamine and subsequent activation with glutaraldehyde as a crosslinking agent. The enzyme load on the textile surface, the activity of the immobilized protein after repeated use as well as the storage stability was evaluated. Scanning electron micrographs and UV Vis spectroscopy made it possible to verify the enzyme immobilization on the textile surface. Furthermore, the load of immobilized peroxidase was determined by ICP OE spectrometry to be 9-12mg per gram of textile. The activity of immobilized Baylase® remained high over 35 reaction cycles and a storage period of 8 weeks.

Microfluidic Reactors Based on Rechargeable Catalytic Porous Supports: Heterogeneous Enzymatic Catalysis via Reversible Host-Guest Interactions.

We report on the fabrication of a microfluidic device in which the reservoir contains a porous surface with enzymatic catalytic activity provided by the reversible immobilization of horseradish peroxidase onto micrometer size pores. The porous functional reservoir was obtained by the Breath Figures approach by casting in a moist environment a solution containing a mixture of high molecular weight polystyrene (HPS) and a poly(styrene-co-cyclodextrin based styrene) (P(S-co-SCD)) statistical copolymer. The pores enriched in CD were employed to immobilize horseradish peroxidase (previously modified with adamantane) by host-guest interactions (HRP-Ada). These surfaces exhibit catalytic activity that remains stable during several reaction cycles. Moreover, the porous platforms could be recovered by using free water-soluble β-CD with detergents. An excess of β-CD/TritonX100 in solution disrupts the interactions between HRP-Ada and the CD-modified substrate thus allowing us to recover the employed enzyme and reuse the platform.

Immobilization and Thermostability of Manganese Peroxidase from Trichoderma harzianum

Immobilization and Thermostability of Manganese Peroxidase from Trichoderma harzianum

Evaluation of a Reduced Graphene Oxide Antimony Nanocomposite Horseradish Peroxidase Biosensor Matrix for Hydrogen Peroxide

An exploration of reduced graphene oxide impregnated with antimony nanoparticles to form a nanocomposite as a platform for the immobilization of horseradish peroxidase for the amperometric detection of hydrogen peroxide is presented. Electrochemical measurements revealed that the heterogeneous electron transfer constant (ks) was evaluated to be 0.37 s−1. The average surface concentration of electro-active HRP on the surface of the modified glassy carbon electrode was calculated to be 9.43 × 10−9 mol cm−2. The voltammetric results revealed that the peak current of the modified electrode increased linearly with increasing concentration of hydrogen peroxide (from 1nM to 4nM) with a detection limit of 2.88nM.

Enhanced Catalytic Activity and Stability of Ginger Peroxidase Immobilized on Amino-Functionalized Silica-Coated Titanium Dioxide Nanocomposite: A Cost-Effective Tool for Bioremediation

This study describes the immobilization of ginger peroxidase on amino-functionalized silica-coated titanium dioxide nanocomposite and its application in bioremediation process. A dramatic enhancement in enzyme activity was observed after immobilization on nanosupport which was evident from the effectiveness factor (η) value of 1.76. Immobilization of enzyme on nanosupport was confirmed by transmission electron microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. V max of the enzyme was changed to 35.01 μmol L−1 min−1 from 8.42 μmol L−1 min−1 after immobilization on nanocomposite, which was a fourfold increase as compared to the free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of the enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process, i.e., 90% of the dye was decolorized within 1.5 h as compared to the free enzyme decolorizing only 69% of the dye in the same period. Our results clearly demonstrate that this nanobioconjugate with enhanced catalytic activity, high stability, and very good reusability has remarkable potential for the treatment of aromatic pollutants present in wastewater.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0mmol/g of dry polymer compared to 1.5mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61mM compared to the soluble enzyme. After 7cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Nickel Oxide Nanoparticles Immobilized on MWCNTs/PANI Composite Film as an Electron Transfer Facilitator for Horseradish Peroxidase: Direct Electron Transfer and H2O2 Determination

In this paper, we developed a new method for immobilization of horseradish peroxidase (HRP) on Nano-NiO/ MWCNTs/PANI composite film, which can effectively promote the direct electron transfer and electrocatalysis of enzyme in neutral solution. This composite film modified electrode was systematically investigated by altering the thickness of composite film, pH value of solution, and the mass ratio of MWCNT to NiO nanoparticles. The study showed that the effective immobilization of horseradish peroxidase on Nano-NiO/ MWCNTs/PANI was 30 cycles of cyclic voltammetry for the preparation of PANI film and the mass ratio of 10:1. The composite film possesses a high catalytic activity in pH 6.8 buffer solution containing H2O2. The plot of the response current to H2O2 concentration exhibited a linear relation at a range of 3.0 × 10−6 to 4.3 × 10−4 M, with a correlation coefficient of 0.998. The detection limit is 4.3 × 10−7 M(S/N = 3). These results indicated that the modified electrode was beneficial to retain the catalytic activity of the enzyme, and possessed good stability and reproducibility.

Amperometric capsaicin biosensor based on covalent immobilization of horseradish peroxidase (HRP) on acrylic microspheres for chilli hotness determination

The evaluation of chilli hotness using amperometric capsaicin biosensor-based enzyme that was immobilized covalently to the surface of modified acrylic microspheres is the first of its kind presented in this work. The immobilization of enzyme covalently to the surface of microspheres via succinimide groups prevented the leaching of the enzyme. The enzymatic reaction between horseradish peroxidase-capsaicin in the presence of hydrogen peroxide, which was mediated by vinyl ferrocene, enabled the current measurement at a low potential (0.22V). Besides, the pungency level of the chilli that was proportional to the capsaicin concentration was measured using this method. This biosensor gave a linear response range towards capsaicin from 0.75–24.94μM (R2=0.992) with a detection limit at 0.39μM. Moreover, the relative standard deviation (RSD) for reproducibility study was 8.2% (n=7). Therefore, this biosensor was successfully applied for evaluation of chilli hotness in chilli sample, as well as in comparison with a standard method that employed the HPLC method.

Enhancement of Peroxidase Stability Against Oxidative Self-Inactivation by Co-immobilization with a Redox-Active Protein in Mesoporous Silicon and Silica Microparticles.

The study of the stability enhancement of a peroxidase immobilized onto mesoporous silicon/silica microparticles is presented. Peroxidases tend to get inactivated in the presence of hydrogen peroxide, their essential co-substrate, following an auto-inactivation mechanism. In order to minimize this inactivation, a second protein was co-immobilized to act as an electron acceptor and thus increase the stability against self-oxidation of peroxidase. Two heme proteins were immobilized into the microparticles: a fungal commercial peroxidase and cytochrome c from equine heart. Two types of biocatalysts were prepared: one with only covalently immobilized peroxidase (one-protein system) and another based on covalent co-immobilization of peroxidase and cytochrome c (two-protein system), both immobilized by using carbodiimide chemistry. The amount of immobilized protein was estimated spectrophotometrically, and the characterization of the biocatalyst support matrix was performed using Brunauer–Emmett–Teller (BET), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared (FTIR) analyses. Stability studies show that co-immobilization with the two-protein system enhances the oxidative stability of peroxidase almost four times with respect to the one-protein system. Thermal stability analysis shows that the immobilization of peroxidase in derivatized porous silicon microparticles does not protect the protein from thermal denaturation, whereas biogenic silica microparticles confer significant thermal stabilization.

Colorimetric sensor based on bubble wrap and camera phone for glucose determination

Glucose is a typical marker of diabetes mellitus but it corresponds also with metabolic syndrome, immune disorders and some types of poisoning. Hence fast, low cost and simple method for its determination is required. Bubble wrap, cheap and available material, was used for immobilization of glucose oxidase (GOx) and peroxidase (POx), the enzymes necessary for colorimetric determination of glucose. Method is based on reaction of enzymes (immobilized in sol-gel membrane inside the bubbles) with substrates glucose and o-phenylenediamine dihydrochloride (o-PD) providing intensive coloration. The color change can be easily tracked by phone integrated camera. Color intensity expressed in red-green-blue (RGB) color model was used for displaying of photos and for gaining numeric data representing concentrations of glucose. The assay exerted good correlation of color intensity with the concentration of glucose, adequately low limit of detection (750 mmol/l) for glucose blood assay, no influence of interferents or matrix substances and by sufficient long term stability of sol-gel membrane. The sensor was found as low-cost simple way to analyze glucose blood levels with promising prospects in the field of portable devices.

Immobilization of horseradish peroxidase on amidoximated acrylic polymer activated by cyanuric chloride

Horseradish peroxidase (HRP) was immobilized on amidoximated acrylic fabric after being activated with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize fabrics. The maximum immobilization efficiency of HRP (70%) was detected at 4% cyanuric chloride and pH 7.0. The immobilized enzyme retained 45% of its initial activity after ten reuses. The immobilization of enzyme on the carrier is saturated after 6h of incubation time. The pH was shifted from 7.0 for soluble HRP to 7.5–8.0 for the immobilized enzyme. The soluble HRP and immobilized HRP had the same optimum activity at 40°C. The immobilized HRP is more thermal stable than soluble HRP. Substrate analogues were oxidized by immobilized HRP with higher efficiencies than those of soluble HRP. Km values of the soluble HRP and the immobilized HRP were 31 and 37mM for guiacol and 5.0 and 7.8mM for H2O2, respectively. The immobilized HRP had higher efficiency for removal of phenol than that of soluble HRP. The immobilized HRP had higher resistance toward heavy metal ions compared to the soluble enzyme. The immobilized HRP was more stable against high concentration of urea, Triton X-100 and isopropanol. The immobilized HRP exhibited high resistance to proteolysis by trypsin than soluble enzyme. In conclusion, the immobilized HRP could be used as a potential efficient catalyst for the removal of aromatic pollutants from wastewater.

Design and Construction of Magnetic Nanoparticles Incorporated with a Chitosan and Poly (vinyl) Alcohol Cryogel and its Application for Immobilization of Horseradish Peroxidase

Magnetic nanoparticles were synthesized by simple co-precipitation method in aqueous medium. The structural characteristics of the powders were studied by XRD and the sizes of nanoparticles were measured with dynamic light scattering. The prepared magnetic nanoparticles were about 43nm in diameter and were then subsequently incorporated into a chitosan and poly (vinyl alcohol) cryogel and applied to immobilize the horseradish peroxidase through covalent binding method (MNP-CS-PVA-HRP cryogel microbar). The MNP-CS-PVA- HRP cryogel microbar was applied for the detection of hydrogen peroxide. The horseradish peroxidase can catalytically oxidize the substrate of hydrogen peroxide and o-dianisidine, generating blown colour that are proportional to the concentrations of hydrogen peroxide. The results showed that the absorption values at 430nm increased with the hydrogen peroxide concentrations ranging from 10-1,000mM. The immobilized horseradish peroxidase on magnetic nanoparticles retained enzyme activity up to the 10th reusable. The proposed approach confirmed that the magnetic nanoparticles not only possessed enzyme activity but also showed potential application in varieties of simple, robust, and cost-effective analytical methods in the future.