Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.     

Removal of linear and branched alkylphenols from aqueous solutions with horseradish peroxidase

Alkylphenols were effectively treated with horseradish peroxidase at pH 7.0 and 30 degrees C in the presence of H(2)O(2) and poly(ethylene glycol) irrespective of the relative position or isomeric form of the alkyl chains. Water-insoluble oligomer precipitates were readily filtered out after enzymatic treatment, and transparent and colorless solutions were obtained for all p- and m-alkylphenols used.     

Partition of horseradish peroxidase in aqueous two-phase systems containing polyvinylpyrrolidone

Growing peroxidase utilisation in different industries encourages the search for high benefit/cost ratio purification methods such as aqueous two-phase partition. In this way, the partitioning behaviour of peroxidase from Armoracia rusticanaroots in polyvinylpirrolidone/Reppal and polyvinylpirrolidone/salt aqueous two-phase systems was investigated. Based on these results, a two-step purification process was developed. In the first system (polyvinylpyrrolidone K30/Reppal PES 200, pH 7.0), cell debris and some contaminating proteins were shifted to the bottom phase while peroxidase concentrated in the top phase. After discarding the bottom phase, the second step involved addition of magnesium sulphate thus forming a second aqueous two-phase system. At this step, the enzyme was extracted into the salt-rich bottom phase. The overall yield was 75% and the purification factor 7.3.This revised version was published online in October 2005 with corrections to the Cover Date.     

Model development for horseradish peroxidase catalyzed removal of aqueous phenol

Once activated by hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the oxidation of aqueous aromatic compounds to produce high molecular weight polymers of low solubility. A pseudo steady-state kinetic model of the HRP-hydrogen peroxide-aromatic compound system was modified to incorporate enzyme inactivation mechanisms in order to improve its predictive ability. The kinetic constants of the model were calibrated using a series of experimental data sets. The model's ability to predict the time-dependent removal of phenol within the range of 0.5-6 mM from a batch reactor was validated. The model accounts for permanent losses of enzyme activity through inactivation by free radicals as well as interaction with end-product polymers as they form. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 251-261, 1997.     

Removal of aqueous pentachlorophenol by horseradish peroxidase in the presence of surfactants

An important issue in the oxidation of pentachlorophenol (PCP) by the enzyme horseradish peroxidase (HRP) is enzyme inactivation during the reaction. This study was initiated to investigate the ability of two nonionic surfactants (Tween 20 and Tween 80) to mitigate HRP inactivation. The surfactants were tested at concentrations below and above their critical micelle concentrations (CMCs). Enhancement of PCP oxidation was observed at sub-CMCs, indicating effective protection of HRP by the two surfactants. Maximum levels of PCP removal were observed when the concentrations of Tween 20 and Tween 80 were 40 and 50% of the CMCs, respectively. At supra-CMCs, both surfactants caused a noticeable reduction in the extent of PCP removal.     

Stabilization of polyacrylamide gel immobilized horseradish peroxidase by its covalent coupling to albumin]

Pretreatment of peroxidase by its covalent coupling to inert proteins and albumin by means of glutaraldehyde considerably increases the thermostability and specific activity of polyacrylamide gel (PAAG) immobilized peroxidase. The effects of PAAG composition on the catalytic properties of the immobilized oligomers: peroxidase-inert proteins-albumin, are studied. The oligomers immobilized in 40% PAAG (10% N,N'-methylenebisacrylamide) possess the maximal specific activity (4.5 nmol/g). The effects of oligomer composition on their catalytic activity and stability in PAAG are studied. The stability of oligomers of optimal composition (ratio of albumin/peroxidase is 2.4), incorporated into 40% PAAG, is 15 times higher as compared to that of the soluble enzyme and 250 times higher as compared to that of the enzyme incorporated into PAAG without pretreatment. A mechanism of stabilizing effect exerted by albumin on peroxidase in PAAG-immobilized oligomers, is discussed.     

辣根过氧化物酶的热稳定剂

保持酶的天然状态和高催化特性具有重要的意义。本研究筛选了辣根过氧化物酶(HRP)的稳定剂并研究了其作用机制。结果发现硫酸镁和明胶能够显著提高HRP的热稳定性,并且两者具有协同作用。在硫酸镁和明胶组成的酶稳定剂存在的条件下,HRP在50oC保温80h后仍能保持89%的活性,常温下存放90d后可保持57%的活性,而未加稳定剂的对照样品中HRP的残留活性分别为6%和小于1%。通过对HRP的Soret带吸收光谱,色氨酸内源荧光,ANS荧光进行分析,揭示酶稳定剂可以明显降低在加热条件下HRP的变性程度,从而维持较为稳定的天然构象。     

Stereospecificity of horseradish peroxidase

We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o-diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (delta1 and delta2). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower Km values for L- than for D-isomers. The kcat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same delta1 and delta2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates (K m) and in their transformation reaction rates (k cat).     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

A study of diluted aqueous solutions of humic acids by scanning microcalorimetry

It has been found by reversed-phase chromatography that humic acids obtained from vermicomposts of different duration of vermicomposting consist of a hydrophilic and a hydrophobic fractions, the hydrophobic fraction having a substantially lower content of charged, probably carboxylic, groups. A change in the sign of the temperature dependence of the heat capacity of diluted aqueous solutions of humic acids at approximately 58 degrees C has been found by differential scanning microcalorimetry, which indicates an increase in the hydration of hydrophobic groups. A jumpwise increase in heat capacity in the temperature range from 86 to 90 degrees C was also found, which is due likely to the hydration of hydrophobic groups in the interior of "micelles", due to the "devitrification" of the hydrophobic nucleus of micelle-like structures. It was shown that increasing the duration of vermicomposting leads to an increase in the relative content of the hydrophobic fraction of humic acids and in the cooperativity of the thermodynamic transition, which manifests itself in a jump of heat capacity, which probably results from the increase in the "micelle" size.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

Chemical stabilization of recombinant horseradish peroxidase

Summary Unglycosylated recombinant horseradish peroxidase (HRP C^*) had a half life of 21 minutes at 65°C compared with only 5 minutes for the plant enzyme (HRP). The half life of HRP C^* at 65°C increased by 5-fold following modification with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Tolerance to 60% 1,4-dioxan also increased whilst tolerance to 30% dimethylformamide was unchanged.     

Calcium-related properties of horseradish peroxidase.

Horseradish peroxidase has been shown to be a metalloprotein in which calcium contributes to the structural stability of the protein. Isoenzyme C and A contain 2.0 and 1.4 moles calcium/mole enzyme, respectively, which can be removed by treatment with guanidine hydrochloride and EDTA. Calcium-free isoenzyme C, but not isoenzyme A, reconstitutes upon addition of calcium and regains enzymatic activity. Free calcium readily exchanges with isoenzyme C, but only to a small extent with isoenzyme A. In addition the role of calcium in maintaining molecular conformation is evidenced by the effects of calcium removal from the isoenzyme C on the thermal stability of the protein.     

Immobilization of horseradish peroxidase onto kaolin

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer–Emmett–Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H₂O₂) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.     

Partition behaviour of horseradish peroxidase isoenzymes in aqueous two-phase poly(ethyleneglycol)/phosphate systems

Summary Partition of purified horseradish peroxidase isoenzymes in aqueous two-phase systems was not affected by pH changes, but tie-line length and NaCl addition greatly increased the partition coefficient of all three isoenzymes, the former having more influence than the latter. In all systems, K were higher for acidic than those for neutral and basic isoenzymes, and K for basic were the lowest.