Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Preparation, morphological characterization, and activity of thin films of horseradish peroxidase

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.     

Silver ion microplates for immunoassays.

Microplate wells can be coated with silver ions using glutaraldehyde as a spacer molecule and thiourea as a complexing ligand. Microwells containing surface silver ions are shown to immobilize biotin-labeled horseradish peroxidase (HRP) in active form, while showing very little affinity for the unlabeled enzyme. These plates can also immobilize biotin-labeled antibodies that exhibit bioactivity after immobilization. Silver ions are needed for the complexation of the biotinylated enzyme or antibody because microwells modified to contain surface amine or thiourea molecules do not immobilize appreciable amounts of the labeled proteins. A maximum surface coverage for biotin-labeled HRP of 40 ng/cm2 and an immobilization binding constant of Km = 8 x 10(9)/M are determined from serial dilutions in a microplate. Detection of as little as 6.7 fmol HRP is achieved using antibodies immobilized on the silver ion-modified microplates. Active antibody surface densities were estimated to be between 130 and 260 nm2/antibody molecule. Background binding of HRP to the modified silver ion microplates was very low, allowing for reasonably accurate detection between 10(-14) and 10(-11) mol HRP.     

Stabilization of activity of oxidoreductases by their immobilization onto special functionalized glass and novel aminocellulose film using different coupling reagents

Glucose oxidase (GOD), horseradish peroxidase (HRP), and lactate oxidase (LOD) were covalently immobilized on special NH(2)-functionalized glass and on a novel NH(2)-cellulose film via 13 different coupling reagents. The properties of these immobilized enzymes, such as activity, storage stability, and thermostability, are strongly dependent on the coupling reagent. For example, GOD immobilized by cyanuric chloride on the NH(2)-cellulose film loses approximately half of its immobilized activity after 30 days of storage at 4 degrees C or after treatment at 65 degrees C for 30 min. In contrast, GOD immobilized by L-ascorbic acid onto the same NH(2)-cellulose film retains 90% of its initial activity after 1 year of storage at 4 degrees C and 92% after heat treatment at 65 degrees C for 30 min. Unlike GOD, in the case of LOD only immobilization on special NH(2)-functionalized glass, e.g., via cyanuric chloride, led to a stabilization of the enzyme activity in comparison to the native enzyme. The operational stability of immobilized HRP was up to 40 times higher than that of the native enzyme if coupling to the new NH(2)-cellulose film led to an amide or sulfonamide bond. Regarding the kinetics of the immobilized enzymes, the coupling reagent plays a minor role for the enzyme substrate affinity, which is characterized by the apparent Michaelis constant (K(M,app)). The NH(2)-functionalized support material as well as the immobilized density of the protein and/or immobilized activity has a strong influence on the K(M,app) value. In all cases, K(M,app) decreases with increasing immobilized enzyme protein density and particularly drastically for GOD.     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and its Electrochemical Properties

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4–9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H₂O₂ but without an added mediator or a reducing substrate.     

Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization

Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer. The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability. The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization. The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI. The storage conditions for PANI/PP membranes containing immobilized HRP were studied. HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5 degrees C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.     

The enzyme coupling process in urease immobilization on O-alkylated nylon tubes

Coupling of Jack bean urease (EC 3.5.1.5) to the inside surface of type 6 nylon tubes, activated by high-temperature O-alkylation with dimethyl sulphate and modified subsequently with lysine and glutaraldehyde, was investigated to establish optimal experimental conditions for the coupling process. For the system described, the most active immobilized urease derivatives were prepared with 2 mg/ml of the solubilized urease solution and use of higher enzyme concentrations proved wasteful. Although urease coupling without thermal denaturation of the solubilized enzyme was achieved at 20 degrees C, derivatives prepared at 37 degrees C yielded maximal activity over the 3 h coupling period. Also, longer incubations of the enzyme solution in the tube were unnecessary under these conditions. Optimal pH for the coupling process was 6.5, one at which the solubilized enzyme was most stable.     

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO₂, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology

The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.