Oxidative Cross-Linking of Pentosans by a Fungal Laccase and Horseradish Peroxidase: Mechanism of Linkage Between Feruloylated Arabinoxylans

The potential of a laccase from the fungus Pycnoporus cinnabarinus to cross-link feruloylated soluble wheat arabinoxylans was investigated using capillary viscometry, size-exclusion HPLC, and reverse-phase HPLC of phenolic compounds. The laccase results were compared with those for a hydrogen peroxide/horseradish peroxidase system. The oxidants provoked an increase in viscosity of a 0.2% (w/v) arabinoxylan solution. A gel was formed after 30 min with laccase. Hydrogen peroxide was consumed rapidly before a gel could be formed. Free ferulic acid, methyl ferulate, and vanillic acid inhibited the gelation, whereas fumaric acid had no effect. This suggests that the aromatic ring, and not the propenoic chain of ferulic acid, was the initiating site for arabinoxylan cross-linking. Ferulic acid and its 8-O-4′, 8-5′, and 5-5′ dehydrodimers were present in nonoxidized arabinoxylans. Upon oxidation, the 8-8′ and 8-5′ benzofuran dehydrodimers appeared and the 8-O-4′ and 8-5′ dimers increased. The production of dimers was proportional to the consumption of ester-bound ferulic acid. In cross-linked arabinoxylans, the major dimers were 8-5′ benzofuran, 8-8′, and 8-O-4′, whereas the 5-5′ dehydrodimer remained at the same level as in the nonoxidized solution.     

Enzymatic oligomerization of vanillic acid

When vanillic acid was incubated with an extracellular laccase from the fungus Rhizoctonia praticola, the formation of various oligomeric products was observed ranging from dimers to pentamers. The three dimers isolated by thin-layer chromatography had molecular ions of 304, 304 and 334, respectively, which corresponded to two dimers (methoxy-p-benzoquinone-vanillic acid) with C-O (2-methoxy-6-(2'-methoxy-4'-carboxyphenoxy)-1,4-benzoquinone) and C-C (2-methoxy-6-(2'-hydroxy-3'-methoxy-5'-carboxyphenyl)-1,4-benzoquinone) coupling, and dehydrodivanillic acid (m/z 334) (2,2'-dihydroxy-3, 3'-dimethoxy-5,5'-dicarboxybiphenyl). The identity of these compounds and their methylated derivatives was confirmed by mass and NMR spectrometric analysis. The oxidative coupling of vanillic acid is also catalyzed by peroxidase but not by tyrosinase.     

Reactivity of ferulic acid and its derivatives toward hydrogen peroxide and peracetic acid.

The reactions of ferulic acid and its derivatives with hydrogen peroxide and peracetic acid in lignin-retaining bleaching conditions have been investigated to determine their susceptibility to oxidative degradation. The conjugated side chain of ferulic acid and its etherified or esterified derivative was shown to be fairly stable, especially to hydrogen peroxide. The major reaction was trans-cis isomerization that possibly involved a radical mechanism but did not cause bond cleavage. The peracetic acid reaction increased the rate of trans-cis isomerization and was also accompanied by a minor cleavage of the side chain. Esterification did not have a substantial effect on the reactivity of ferulic acid, but 4-O-etherification significantly stabilized it against these two oxidants. By contrast, aldehyde substitution tremendously enhanced the susceptibility of the cinnamyl side chain to oxidative degradation, as evidenced by an intensive degradation of coniferaldehyde.     

Broccoli processing wastes as a source of peroxidase

A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems ( Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 x 10(6) M(-1) s(-1). The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 degrees C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification.     

Covalent binding of the flavonoid quercetin to human serum albumin

Quercetin is an abundant flavonoid in the human diet with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be important for its biological activities. In the present study, using radiolabeled drug and quantifying the products after electrophoretic separation, proteins to which oxidized quercetin is binding irreversibly were identified. The binding of quercetin to human serum albumin (HSA) in human blood and the effect of stimulation of neutrophilic myeloperoxidase on this binding were also measured. The in vitro binding of quercetin to eight proteins in the presence of catalytic amounts of horseradish peroxidase and hydrogen peroxide was highly selective for HSA. For all proteins the binding was dramatically decreased by reduced L-glutathione. In the blood samples, the release of neutrophilic myeloperoxidase by phorbol ester caused a 3-fold increase in the binding of quercetin to HSA. This study shows that quercetin in the presence of peroxidase/hydrogen peroxide covalently links to proteins with a particularly high affinity for HSA and that this also may occur in vivo after exposure to quercetin. This provides further insights into the complex behavior of this major dietary flavonoid.     

Analysis and modeling of the ferulic acid oxidation by a glucose oxidase-peroxidase association. Comparison with a hexose oxidase-peroxidase association

A commercial glucose oxidase (GOX) from Aspergillus niger was partially characterized. The enzyme exhibited a two-step transfer mechanism, and the kinetic constants toward glucose and oxygen were determined. Under conditions similar to dough making (glucose concentration and pH), GOX does not exhibit maximum activity. A hexose oxidase (HOX) from Chondrus crispus was partially characterized as well. The HOX activity is not far from the optimum in the kneading conditions (pH and glucose concentration). A peroxidase (POD) purified from wheat germ was used to oxidize ferulic acid in the presence of GOX or HOX. Hydrogen peroxide produced during the glucose oxidation activates the wheat germ POD. Ferulic acid oxidation in solutions containing different ratios of POD + GOX or HOX + POD was followed by UV spectrophotometry. For the same dosage, the HOX-POD system is the most efficient for peroxidase activation. Using absorbance data and kinetic constants of GOX and POD, a mathematical model describing the release or consumption of the different reactants (hydrogen peroxide, oxygen, and ferulic acid) in the medium was developed, and experimental data correlated well with calculated values. The results obtained will be applied to investigate the effect of GOX and HOX activities on the rheological properties of dough.     

Kinetics of enzyme-catalyzed cross-linking of feruloylated arabinan from sugar beet

Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.     

Novel oxidations of (+)-catechin by horseradish peroxidase and laccase

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.     

Oxidation of ferulic acid or arabinose-esterified ferulic acid by wheat germ peroxidase

The oxidation of ferulic acid (FA) or 5-O-(trans-feruloyl)-L-arabinose (EFA) by a purified wheat germ peroxidase was followed by UV spectrophotometry and high-performance liquid chromatography using an electrochemical detection. Wheat peroxidase (POD) exhibits a ping-pong bireactant mechanism forming phenoxy radicals more rapidly from FA than from EFA in routine assay conditions. When both the free and the esterified forms of FA are present, the reverse was found. This result could be due to a nonenzymatic cooxidation of FA by the phenoxy radicals of EFA leading to the formation of phenoxy radicals of FA and the EFA regeneration. Addition of ascorbic acid (AA) provokes a delay of FA consumption. AA reduced very rapidly the phenoxy radicals formed by POD back to initial phenol avoiding the formation of ferulate dimers until it was completely oxidized in dehydroascorbic acid. Conversely, cysteine addition slowed but did not delay the FA consumption. The thiol reduced a fraction of the phenoxy radicals produced by wheat POD and was oxidized into cystine, while the other part of phenoxy radicals formed ferulate dimers. These results could be of interest to understand the POD effect on the wheat dough rheological properties.     

Enzymatic oxidative treatments of wheat bran layers: effects on ferulic acid composition and mechanical properties

Enzymatic treatments known to induce the gelation of feruloylated arabinoxylans solutions were applied to tissue strips isolated from peripheral layers of wheat grain to tentatively produce in situ arabinoxylan reticulation. The treatments by horseradish peroxidase (HRP) and manganese dependent peroxidase (MnP) induced a dimerization of ferulic acid (FA) in wheat bran with concomitant decrease of arabinoxylan solubility. Similar results were obtained, but to a lesser extent, by simple incubation of bran strips in water, suggesting the action of endogenous peroxidases. The fact that these treatments proved to be ineffective on the isolated aleurone layer and pericarp suggested that dimerization occurred mostly at the aleurone-pericarp interface. In addition, the MnP system generated a consumption of monomer and dimer of ferulic acid in the pericarp, perhaps due to their incorporation into lignin. Micro-mechanical tests using DMTA were performed on isolated tissue strips and showed that oxidation of wheat bran increased their mechanical strength (increase of stress and strain to rupture).     

Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.     

Novel tripeptides with α-glucosidase inhibitory activity isolated from silk cocoon hydrolysate

Active compounds with antidiabetic potential were isolated from silk peptide E5K6 by consecutive ultrafiltration and gel filtration using Biogel P-2 and RS-HPLC using a YMC-Pack Pro C18 column. The highest α-glucosidase inhibitory activity of silk peptide E5K6 resulted from fractions with MW <1 kDa. The activities of gel-filtered fractions from silk peptide E5K6 of <1 kDa were assayed in vitro, demonstrating that the fourth peak (F4) had the highest α-glucosidase inhibitory activity (IC(50) = 37.1 mg/mL). F4 of silk peptide E5K6 was separated by HPLC into two peaks. Moreover, the purified compounds were identified as Gly-Glu-Tyr (GEY, MW = 367 Da) and Gly-Tyr-Gly (GYG, MW = 295 Da) according to amino acid sequences, and their α-glucosidase inhibitory activities (IC(50)) were 2.7 and 1.5 mg/mL, respectively.     

Development and Application of a Methodology to Determine Free Ferulic Acid and Ferulic Acid Ester‐Linked to Different Types of Carbohydrates in Cereal Products

Ferulic acid bioavailability is dependent on its form present in food. This necessitates a methodology to quantify different groups of ferulic acid derivatives in food products, especially cereal‐based products. The aim of the proposed methodology is to separate and quantify ferulic acid ester‐linked to mono‐ and/or oligosaccharides (OF), to soluble polysaccharides (SPF), and to insoluble polysaccharides (IPF) as well as in its free form. Development and partial validation of this method, which was widely based on liquid/liquid extraction and precipitation steps, was performed using characterized standard materials isolated from corn bran. As the determination of OF was one of the major goals of this methodology, three different feruloylated mono‐ and oligosaccharides were used for method development and validation. To determine the accuracy of the method, ferulic acid–containing standard materials added to a starch matrix were extracted and separated according to the developed protocol. The separated ferulic acid esters were saponified before ferulic acid was analyzed by reversed phase HPLC. Recovery rates were generally between 70 and 103%, with the lowest recovery rates for SPF and highest recovery rates for IPF and OF. Finally, the applicability of the method to unprocessed and processed wheat bran samples was demonstrated.     

Soybean peroxidase-catalyzed oxidation of luminol by hydrogen peroxide

Anionic soybean peroxidase Glycine max (SbP) is shown to efficiently catalyze luminol oxidation by hydrogen peroxide. Contrary to horseradish peroxidase, the presence of p-iodophenol in the reaction medium affects slightly the efficiency of SbP catalysis. A maximal intensity of chemiluminescence, produced through this enzymatic reaction, was detected at pH 8.4-8.6. Contrary to anionic palm tree peroxidase, in the presence of SbP, chemiluminescence intensity increases with the reaction buffer concentration. The detection limit of SbP in the reaction of luminol oxidation is 0.3 x 10(-12) M. Therefore, high sensitivity in combination with the long-term chemiluminescent signal is indicative of good prospects for application of this enzyme in enzyme immunoassay with chemiluminescent detection.     

玉米铬毒性研究：氧化损伤、渗透调节物质积累、抗氧化防御以及铬的吸收

Agricultural production systems are immensely exposed to different environmental stresses in which heavy metal stress receives serious concerns. This study was conducted to explore the deleterious effects of different chromium(Cr) stress levels, i.e., 0, 30, 6090, 120, and 150 μmol L-1, on two maize genotypes, Wandan 13 and Runnong 35. Both genotypes were evaluated by measuring their growth and yield characteristics, Cr accumulation in different plant tissues, alterations in osmolyte accumulation, generation o reactive oxygen species(ROS), and anti-oxidative enzyme activity to scavenge ROS. The results showed that Cr stress decreased the leaf area, cob formation, 100-grain weight, shoot fresh biomass, and yield formation, while Cr accumulation in different maize tissues was found in the order of roots leaves stem seeds in both genotypes. The increased Cr toxicity resulted in higher free proline soluble sugars and total phenolic contents, and lower soluble protein contents. However, enhanced lipid peroxidation was noticed in the forms of malondialdehyde, hydrogen peroxide(H2O2) and thiobarbituric acid reactive substance accumulation, and electrolyte leakage. The hyperactivity of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, especially glutathione peroxidase and glutathione reductase indicated that these anti-oxidative enzymes had a central role in protecting maize from Cr toxicity, especially for Wandan 13. Moreover, higher uptake and less translocation of Cr contents into the grains of Wandan 13 implied its importance as a potential candidate against soil Cr pollution.     

Effect of cysteinyl caffeic acid, caffeic acid, and L-dopa on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase

To study a way to covalently link arabinoxylans and proteins using a fungal laccase from the fungus Pycnoporus cinnabarinus, the effect of cysteinyl caffeic acid on the cross-linking of wheat arabinoxylans was investigated by means of capillary viscometry and RP-HPLC of alkali labile phenolic compounds. Cysteinyl caffeic acid provoked a delay in gelation and in the consumption of the esterified ferulic acid on arabinoxylans. When reacting free ferulic acid and cysteinyl caffeic acid with laccase, the ferulic acid consumption and the dehydrodimers production were also diminished. These results suggest that cysteinyl caffeic acid is oxidized while reducing the semiquinones of ferulic acid produced by laccase. Thus, ferulic acid could not be oxidized into dimers until all cysteinyl caffeic acid was consumed, preventing the cross-linking of feruloylated arabinoxylan chains. A similar mechanism is proposed in the case of caffeic acid and of L-Dopa.     

Degradation of the coffee flavor compound furfuryl mercaptan in model Fenton-type reaction systems

The stability of the coffee flavor compound furfuryl mercaptan has been investigated in aqueous solutions under Fenton-type reaction conditions. The impact of hydrogen peroxide, iron, ascorbic acid, and ethylenediaminetetraacetic acid was studied in various combinations of reagents and temperature. Furfuryl mercaptan reacts readily under Fenton-type reaction conditions, leading to up to 90% degradation within 1 h at 37 degrees C. The losses were lower when one or more of the reagents was omitted or the temperature decreased to 22 degrees C. Volatile reaction products identified were mainly dimers of furfuryl mercaptan, difurfuryl disulfide being the major compound. In addition, a large number of nonvolatile compounds was observed with molecular masses in the range of 92-510 Da. The formation of hydroxyl and carbon-centered radicals was indicated by electron paramagnetic resonance spectra using alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide as spin traps. Whereas *OH was generated by Fenton-type reactions, the C-centered radical is probably a secondary product of the reaction of *OH with various organic molecules, the reaction with furfuryl mercaptan appearing to be the most important. No evidence for S-centered radicals was seen in the spin-trapping experiments, but a sulfur-containing radical was detected when measurements were made at 77 K in the absence of spin traps.     

Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with beta-casein

Heterologous conjugates of wheat arabinoxylan and beta-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of beta-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the beta-casein and the feruloylated arabinoxylan was reached at a protein-to-arabinoxylan ratio of 10:1, in combination with a molar ratio hydrogen peroxide to substrate of 2:1 and a molar protein-to-enzyme ratio between 10(2) and 10(4). The protein-arabinoxylan adducts were separated from the arabinoxylan homopolymers by size exclusion and anion exchange chromatography. The molar ratio protein:arabinoxylan in the purified conjugates varied between 0.1 and 5.6. This is the first report on the large-scale enzymatic preparation of heterologous protein-arabinoxylan conjugates.     

Deodorization of swine manure using minced horseradish roots and peroxides

Public concerns about offensive odors from livestock manures are on the rise and so is the pressure to develop practical ways to reduce the odors. The use of minced horseradish (Armoracia rusticanaL) roots (1:10 w/v plant tissue to swine slurry ratio), with calcium peroxide (CaO2 at 26 or 34 mM) or hydrogen peroxide (H2O2 at 34, 52, or 68 mM) for the deodorization of swine manure, was evaluated through a series of laboratory experiments. The principle underlying this deodorization method is the oxidation of odorants by the concerted action of horseradish peroxidase (present in the plant tissue) and peroxide that serves as an electron acceptor, followed by polymerization of phenolic odorants with a possible copolymerization or adsorption of other odorant compounds. The deodorization effect was assessed by a human panel and gas chromatography (GC). In the case of the GC method, 12 compounds commonly associated with malodor (7 volatile fatty acids or VFAs, 3 phenolic compounds, and 2 indolic compounds) were used as odor indicators. Malodor assessment of the treated slurry by a human panel indicated a 50% reduction in odor intensity. GC results showed 100% removal of all phenolic odorants without reoccurrence for at least 72 h. In view of these data, using plant materials as enzyme carriers and peroxides as electron acceptors emerges as an effective approach to phenolic odor control in animal manure.     

Purification and kinetic characterization of an anionic peroxidase from melon (Cucumis melo L.) cultivated under different salinity conditions

The partial characterization of an anionic peroxidase in melon fruit is described. Four melon peroxidase (MPX) isoenzymes were detected in crude extracts after isoelectric focusing. The major MPX isoenzyme (pI = 3.7) was partially purified by including hydrophobic and anion-exchange chromatography in the purification scheme. The sample obtained was used to characterize MPX. This peroxidase did not show activity on ascorbic acid but oxidized guaiacol at a high rate, showing an optimum pH of 5.5 when acting on this last reducing substrate. Melon fruits grown under highly saline conditions showed slightly increased levels of this anionic isoenzyme. Kinetic studies using 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) as reducing substrate showed that increased salinity in the growth medium did not modify the kinetic parameters of melon peroxidase on both hydrogen peroxide and reducing substrate.