Benzo[a]pyrene degradation using simultaneously combined chemical oxidation,biotreatment with Fusarium solani and cyclodextrins

The interest of simultaneously combining chemical (Fenton’s reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5 × 10⁻³ M H₂O₂ as Fenton’s reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5 × 10⁻³ M hydroxypropyl-β-cyclodextrin (HPBCD) in comparison with randomly methylated-β-cyclodextrin (RAMEB). When Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation.     

The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases

The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H₂O₂ and Fe²⁺ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H₂O₂/Fe²⁺ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H₂O₂/Fe²⁺ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased. Received: 23 December 1996 / Received revision: 17 April 1997 / Accepted: 19 April 1997     

Generation of hydrogen peroxide by brown adipose tissue mitochondria

This is the first report on the generation of H₂O₂ by brown adipose tissue mitochondria. Flavin dehydrogenase-linked substrates like succinate, glycerol-1-phosphate, and fatty acyl CoA were good substrates for the reaction, while NAD⁺-linked substrates were less effective. In cold-acclimated animals the activity showed a substantial increase (2.5-fold). TheK _m andV _max of the reaction were considerably lower than those of the respective dehydrogenase. Metal ions, particularly Cu²⁺ and Fe²⁺ were potent inhibitors of the reaction. Nucleoside diphosphates, which were inhibitors by themselves, potentiated the inhibitory action of Fe²⁺ ions. In most of the properties, the H₂O₂ generator of brown adipose tissue mitochondria resembled that of liver mitochondria.     

Effect of Modified Fenton’s Reaction on Microbial Activity and Removal of PAHs in Creosote Oil Contaminated Soil

This study describes the removal of polycyclic aromatic hydrocarbons (PAHs) from creosote oil contaminated soil by modified Fenton’s reaction in laboratory-scale column experiments and subsequent aerobic biodegradation of PAHs by indigenous bacteria during incubation of the soil. The effect of hydrogen peroxide addition for 4 and 10 days and saturation of soil with H₂O₂ on was studied. In both experiments the H₂O₂ dosage was 0.4 g H₂O₂/g soil. In completely H₂O₂−saturated soil the removal of PAHs (44% within 4 days) by modified Fenton reaction was uniform over the entire soil column. In non-uniformly saturated soil, PAH removal was higher in completely saturated soil (52% in 10 days) compared to partially saturated soil, with only 25% in 10 days. The effect of the modified Fenton’s reaction on the microbial activity in the soil was assessed based on toxicity tests towards Vibrio fischeri, enumeration of viable and dead cells, microbial extracellular enzyme activity, and oxygen consumption and carbon dioxide production during soil incubation. During the laboratory-scale column experiments, the toxicity of column leachate towards Vibrio fischeri increased as a result of the modified Fenton’s reaction. The activities of the microbial extracellular enzymes acetate- and acidic phosphomono-esterase were lower in the incubated modified Fenton’s treated soil compared to extracellular enzyme activities in untreated soil. Abundance of viable cells was lower in incubated modified Fenton treated soil than in untreated soil. Incubation of soil in serum bottles at 20 °C resulted in consumption of oxygen and formation of carbon dioxide, indicating aerobic biodegradation of organic compounds. In untreated soil 20–30% of the PAHs were biodegraded during 2 months of incubation. Incubation of chemically treated soil slightly increased PAH-removal compared to PAH-removal in untreated soil.     

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel. In monocytes/macrophages, H₂O₂-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H₂O₂ are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H₂O₂ were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H₂O₂-induced TRPM2-mediated increase in intracellular free Ca²⁺ ([Ca²⁺]_i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H₂O₂-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe²⁺-accumulation following pretreatment with FeSO₄. Thus intracellular Fe²⁺-accumulation sensitizes H₂O₂-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe²⁺-accumulation increased poly(ADP-ribose) levels in nuclei by H₂O₂ treatment, and the sensitization of H₂O₂-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe²⁺-accumulation enhances H₂O₂-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO₄ stimulated H₂O₂-induced TRPM2 activation at 37 °C in U937 cells and enhanced H₂O₂-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H₂O₂ to cells under conditions of intracellular Fe²⁺-accumulation caused cell death, concentration of H₂O₂ required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe²⁺-accumulation sensitizes TRPM2 channels to H₂O₂ and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H₂O₂-induced TRPM2 channels by Fe²⁺ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe²⁺ is released by heme degradation under intracerebral hemorrhage.     

Light generation with Fenton's reagent its relationship to granulocyte chemiluminescence

A simple chemical system consisting of FeSO₄ and H₂O₂ (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H₂O₂ was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH^.) and singlet oxygen (¹Δ_gO₂) quenchers suppressed chemiluminescence of the FeSO₄ + tryptophan + H₂O₂ system; and, deuterium oxide (²H₂O) enhanced chemiluminescence of both FeSO₄ reactions. These observations suggest that a radical chain reaction involving both OH^. and ¹Δ_gO₂ is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H₂O₂. Since iron and H₂O₂ are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Enhanced transformation of polycyclic aromatic hydrocarbons using a combined Fenton's reagent,microbial treatment and surfactants

The potential for using Fenton's reagent (H₂O₂ + Fe²⁺) as an advanced oxidation pretreatment process to enhance microbial transformation of two model polycyclic aromatic hydrocarbons, anthracene and benzo[a]pyrene, in an aqueous system was evaluated. Fenton's reagent at a concentration of 0.5% H₂O₂ and 10 mM Fe²⁺ (molar ratio, 15:1) was most effective in transforming anthracene at pH 4. Application of non-ionic surfactants during Fenton's pre-treatment was found to be more effective in the transformation of both anthracene and benzo[a]pyrene. The extent of removal of substrates by a combined Fenton's–biotreatment was 2–4 times higher than with Fenton's treatment or biotreatment alone. In a chemical–biological treatment train, 48 h of Fenton's pre-treatment in the presence of a non-ionic surfactant, followed by 7 days of biological treatment resulted in 80–85% removal of PAHs (100 ppm). Electronic Publication     

Suicide-Peroxide inactivation of microperoxidase-11: A kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H₂O₂. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H₂O₂]>>[AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, α_i, and the apparent inactivation rate constant, k_i, were obtained as 0.282 ± 0.006 min^{? 1} and 0.497 ± 0.013 min^{? 1} at [H₂O₂] = 1.0 mM, 27°C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Inactivation of human myeloperoxidase by hydrogen peroxide

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H₂O₂ in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10⁻³ s⁻¹. Treatment of MPO with high H₂O₂ concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H₂O₂ is proposed to occur via the generation of reactive oxidants when H₂O₂ reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.     

Purification of polyhydroxybutyrate produced by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> IPT64 through a chemical and enzymatic route

This work aimed at the study of purification of biopolymers produced by Burkholderia cepacia IPT64, through a chemical attack like an adjuvant procedure through chemical and enzymatic route. Enzymatic lysis using protease was run on an enzyme/cell ratio of 0.02. Chemical attacks were performed as pre or post-treatment for enzymatic attacks, using sodium dodecyl sulphate or hydrogen peroxide. The same chemicals and procedures were used alone as a control. Fenton’s reagent was also tested as a chemical treatment. Using only one of the chemicals H₂O₂, Fenton’s reagent or SDS, the purity increase achieved values of 14%, 16% or 23%, respectively. When H₂O₂ or SDS were used as pre-treatment for the enzymatic attack, the results of purity increase achieved values of 58% for H₂O₂/cell ratio between 0.60 and 1.20, and 57% when SDS/cell ratio at 0.56 was used. In the case when H₂O₂ or SDS were used as post-treatment for the enzymatic attack, results of purity increase achieved 60% when the H₂O₂ was used at H₂O₂/cell ratio ranging between 0.30 and 0.60 and 71% when SDS was used at a ratio SDS/cell of 0.56.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N₂O (2NO + 2e⁻ + 2H⁺ → N₂O + H₂O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O₂-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O₂, we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O₂ resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O₂ shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O₂ as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O₂ show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Ädelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the fenton reagent (Fe2+/H2O2): Nonradicalvs. Radical-induced oxidation

In the absence of added Fe²⁺, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP _i per mg protein per min) is moderately inhibited by H₂O₂ in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H₂O₂. The process, probably a direct oxidative action of H₂O₂ on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both theK _m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/L Fe²⁺ +20 mmol/L H₂O₂), which causes a fast production of HO⁻ radicals, the ATPase is 50–60% inactivated and 90% of added Fe²⁺ is oxidized to Fe³⁺ within 1 min. The inactivation occurs only when Fe²⁺ is added before H₂O₂ and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO⁻ radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe²⁺ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe²⁺ for H₂O₂, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO⁻ production.     

Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO₂, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe²⁺, α-ketoglutarate, O₂ and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO₂, succinate and Fe²⁺, in which Fe²⁺ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO₂ is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO₂ and succinate or after the release of all products, including Fe²⁺, and dehydroascorbate is released before the binding of Fe²⁺. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical.Zn²⁺, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe²⁺, α-ketoglutarate, O₂ and ascorbate respectively, and epinephrine noncompetitively with regard to all cosubstrates. Apparent K_i values are given for the product and other inhibitors.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe₃O₄–chitosan (Fe₃O₄–CS) nanoparticles as support. The magnetic Fe₃O₄–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe₃O₄ nanoparticles made through co-precipitation of Fe²⁺ and Fe³⁺. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe₃O₄–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

Degradation and Mineralization of Phenol Compounds with Goethite Catalyst and Mineralization Prediction Using Artificial Intelligence

The efficiency of phenol degradation via Fenton reaction using mixture of heterogeneous goethite catalyst with homogeneous ferrous ion was analyzed as a function of three independent variables, initial concentration of phenol (60 to 100 mg /L), weight ratio of initial concentration of phenol to that of H₂O₂ (1: 6 to 1: 14) and, weight ratio of initial concentration of goethite catalyst to that of H₂O₂ (1: 0.3 to 1: 0.7). More than 90 % of phenol removal and more than 40% of TOC removal were achieved within 60 minutes of reaction. Two separate models were developed using artificial neural networks to predict degradation percentage by a combination of Fe³⁺ and Fe²⁺ catalyst. Five operational parameters were employed as inputs while phenol degradation and TOC removal were considered as outputs of the developed models. Satisfactory agreement was observed between testing data and the predicted values (R² _Phenol = 0.9214 and R²TOC= 0.9082).