辣根过氧化物酶模拟酶研究进展

辣根过氧化物酶 (HRP)是一种常用的工具酶 ,对其模拟酶的研究是近年来生物化学和有机化学的重要课题 ,具有重要的理论意义和应用价值。本文评述了近十年来HRP模拟酶的研究进展。     

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques.     

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.     

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Tomato peroxidase: purification, characterization, and catalytic properties

A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.     

Heterologous Expression,Purification and Characterization of a Peroxidase Isolated from <Emphasis Type="Italic">Lepidium draba</Emphasis>

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.     

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.     

Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme

A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli. The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme. HRP C* was solubilized and conditions under which it folded to give active enzyme were determined. Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione. Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C. Glycosylation is thus not essential for correct folding and activity. The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding. However, Ca2+ appears to play a key role in folding to give the active enzyme. The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.     

Horseradish peroxidase—catalyzed hydroxylation of phenol: I. Thermodynamic analysis

We analyzed the horseradish peroxidase (HRP)—catalyzed hydroxylation of phenol in the presence of dihydroxy-fumaric acid and oxygen. All of the intermediate forms of the enzyme are reviewed. The last step of hydroxylation, consisting of the production of OH^• radicals that further react on phenol, is emphasized. Possible OH^• radicals production reactions were compiled and analyzed with respect to the available thermodynamic data. Some results of electrochemical experiments were also used to choose the correct set of reactions. At the end of analysis only two reactions for producing OH^• seemed to be consistent with the thermodynamic and experimental data. Neither of these reactions involved compound III or any other intermediate form of HRP. The last step of hydroxylation was thus totally independent of the pure catalytic cycle of the enzyme. As a consequence, HRP cannot be used as an hydroxylation enzyme in place of the P₄₅₀ cytochrome, as is sometimes suggested.     

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.     

Enzymatic detection of native and derivatized horseradish peroxidase in sodium dodecyl sulfate polyacrylamide gels

We developed procedures for the restoration of peroxidatic activity in native horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) following electrophoresis in SDS-polyacrylamide gels (SDS-PAGE). After extraction of SDS with isopropanol from gels containing HRP and WGA-HRP, the peroxidatic activity in these probes could be demonstrated by tetramethylbenzidine (TMB) chemistry. This procedure also showed HRP enzyme activity in electrophoresed tissue homogenates containing HRP. Both free HRP as well as WGA-HRP preparations contain several molecular weight species that display peroxidatic activity. These findings are important for cell biological studies utilizing these substances as molecular probes. The procedures described here should be useful for the analysis of the enzymatically active molecular forms of these frequently used markers in vitro and in vivo.     

Mediated electrochemistry of peroxidases-effects of variations in protein and mediator structures

The kinetics of a range of ferrocene derivatives with horseradish peroxidase (HRP), cytochrome c peroxidase (CCP) and 3 charge reversal mutants of cytochrome c peroxidase were measured using cyclic voltammetry. Substantial differences in rate constant (100 fold) were observed between HRP and CCP for the same mediator with smaller differences (4–5 fold) for different mediators with the same enzyme. The rate constant did not seem to be dependent on redox potential differences. Cluster analysis is proposed as a way of classifying mediator reactivity.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

A novel and efficient oxidative functionalization of lignin by layer-by-layer immobilised Horseradish peroxidase

Horseradish peroxidase (HRP) was chemically immobilised onto alumina particles and coated by polyelectrolytes layers, using the layer-by-layer technique. The reactivity of the immobilised enzyme was studied in the oxidative functionalisation of softwood milled wood and residual kraft lignins and found higher than the free enzyme. In order to investigate the chemical modifications in the lignin structure, quantitative (31)P NMR was used. The immobilised HRP showed a higher reactivity with respect to the native enzyme yielding extensive depolymerisation of lignin.     

BSA和PEG影响固定化辣根过氧化物酶HRP在有机相中活力

ＢＳＡ和ＰＥＧ可以有效地提高固定化辣根过氧化物酶（ＨＲＰ）在有机相中的活力。固定化酶活力的提高与试剂加入的顺序有密切的联系;不同载体对酶的影响不同,Ｇｅｌｉｔｅ,ａｌｕｍｉｎａ,ＸＡＤ－７,Ｋｉｓｅｌｇｅｌ和Ｆｌｏｒｉｓｉｌ为载体,分别以吸附法制备固定化酶。实验表明固定化过程中保护剂和酶的加入顺序与国家化酶活力密切相关,而这些载体的固定化效果又以Ｃｅｌｉｔｅ最佳,Ｆｌｏｒｉｓｉｌ最差。Ｆｌｏｒｉｓ     

An efficient methodology for the purification of date palm peroxidase: Stability comparison with horseradish peroxidase (HRP)

In the present study, Peroxidase from date palm (Phoenix dactylifera) leaves was purified to homogeneity by three-step procedure including aqueous two-phase system, hydrophobic and Ion-exchange chromatography. The enzyme migrated as single band on SDS-PAGE giving molecular weight of 68?±?3?kDa. The purification factor for purified date palm peroxidase was 68 with high 41% yield. Enzymatic assays together with far-UV circular dichroism (CD), intrinsic and extrinsic fluorescence studies were carried out to monitor the structural stability of date palm and horseradish peroxidase (HRP) against various pH and temperatures. Activity measurements illustrated different pH stability for date palm and HRP. Both peroxidases are more susceptible to extreme acidic conditions as suggested by 4 & 15?nm red shift in date palm and HRP, respectively. Secondary structure analysis using far UV-CD exhibited predominance of α-helical (43.8%) structure. Also, pH induces loss in the secondary structure of date palm peroxidase. Thermal stability analysis revealed date palm peroxidase is more stable in comparison to HRP. In summary, date palm peroxidases could be promising enzymes for various applications where extreme pH and temperature is required.     

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.     

Enhanced intracellular stability of dextran-horse radish peroxidase conjugate: an approach to enzyme replacement therapy.

Horse radish peroxidase (HRP), a mannose-containing glycoprotein was covalently modified by conjugation with dextran. The rapid uptake of HRP by the liver is markedly inhibited by mannan. The uptake of dextran-HRP conjugate by the liver, though lower compared to that of the free enzyme, is also partially inhibited by mannan. Liposomes were therefore used as carriers for delivering the free and the modified HRP to the liver. The dextran-HRP conjugate showed greater stability intracellularly as compared to the free enzyme. The enhanced stability of enzymes upon their extensive glycosylation with nondegradable sugar polymers would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of certain enzyme deficiency disorders.     

Extraction of horseradish peroxidase and its conjugates formed by surface-active agent micelles

The author studied peculiarities of the extraction of horseradish peroxidase (HRP) and its conjugates with 3 and 7 molecules of progesterone (PROG) from the aqueous solution into heptane and chloroform containing reversed micelles of surfactants. Micelles of cetyltrimethylammonium bromide, Aerosol OT, and Triton X-45 protect the enzyme from denaturation in the biphasic system. The enzyme is readily extracted from the aqueous phase in the organic medium containing reversed micelles of surfactants at low values of pH. The addition of PEG-6000 (5%) to the aqueous phase enhances the enzyme solubilization at pH 8.6-9.0. The enzyme solubilization significantly increases, when surfactants with unlike charges are used. Inorganic salts decrease the specific solubilization of the enzyme. The HRP modification with progesterone has a weak effect on the enzyme solubilization with reversed micelles.