Presence Of Thiol Compounds Research Articles

Gold nanoclusters-decorated graphitic carbon nitride nanocomposites with antibacterial and anti-biofilm activities

Gold nanoclusters (AuNCs) are of great potential in antibacterial field, but their unsatisfactory stability as a result of high surface free energy limits their practical applications. One efficient solution is loading the ultrasmall AuNCs onto a substrate or matrix. In this work, graphitic carbon nitride (CN) nanoparticles were firstly functionalized with imidazole groups via a photocatalytic reaction. Then, AuNCs were in-situ synthesized on surface of CN by using imidazole moieties as the reducing and stabilizing ligands via formation of Au-N bonds. Transmission electron microscopy (TEM) analysis of the obtained CN-AuNC nanocomposites verified the uniform distribution of AuNCs with a mean size of 1.8 nm on CN surface. Since the coordination strength of Au-N bond is weaker than that of Au-S bond, ligand exchange can occur for the imidazole-protected AuNCs due to ligand exchange in the presence of thiol compounds. Accordingly, the obtained CN-AuNC nanocomposites exhibited thiol depletion ability. The AuNCs slowly released from the CN surface can enter Escherichia coli (E. coli) due to their ultrasmall size, consume bio-thiols reducing species and increase the oxidative stress, which effectively killed the bacteria and inhibited subsequent biofilm formation. This paper provides a promising bactericidal nanocomposite with intrinsic properties for sterilization.

Imidazole-stabilized gold nanoclusters with thiol depletion capacity for antibacterial application

Surface ligand plays a crucial role in regulating the physicochemical properties and functionalities of gold nanoclusters (AuNCs). Compared to the widely studied thiolate-stabilized AuNCs, the reports on imidazole-based ligands to protect AuNCs are still rare. Herein, we report the facile preparation of imidazole-stabilized AuNCs using 1-vinylimidazole as both a reducing and capping agent. The coordination pattern of imidazole ring to Au core was proved by formation of Au-N bond, which can protect the AuNCs with moderate stability against different pH and temperature. Owing to the weaker binding strength of Au-N bond than Au-S bond, the obtained AuNCs possessed thiol depletion capacity due to ligand exchange in the presence of thiol compounds. By consuming the cellular bio-thiols that act as the reducing species in redox balance, the imidazole-protected AuNCs can cause the accumulation of oxidizing species and the elevation of the oxidative stress. Accordingly, the obtained AuNCs exhibited intrinsic antibacterial activity toward Staphylococcus aureus, which could be a promising bactericidal nanomaterial for sterilization.

Thiol Compounds, Pre-Conditioning and Orientation of Explants – Important Factors Affecting Regeneration from Cotyledons of Legume Crop Sesbania Aculeata

Abstract Sesbania aculeata is a multipurpose legume crop grown primarily for green manuring in the rice-based cropping system. Besides this, it is an industrial crop and is also used as food in many parts of the world. The present work reports for optimization of various parameters (growth medium, plant growth regulators, pre-conditioning, orientation of explant, and presence of thiol compounds) affecting in vitro regeneration using mature cotyledon explants. The 5-day-old mature cotyledon explants excised from seedlings grown on Murashige and Skoog (MS) salts and Gamborg (B5) vitamins medium containing 15 μM 6-benzylaminopurine were cultured with its adaxial side facing on medium containing 2.5 μM 6-benzylaminopurine and 50 mg/L thiourea and produced multiple shoots (7 ‒ 8) in 100% cultures within 28 days. Healthy shoots were rooted on half-strength Murashige and Skoog (MS) salts and full-strength vitamins medium augmented with 2.5 μM indole-3-butyric acid.

5′-DMT-protected double-stranded DNA: Synthesis and competence to enzymatic reactions

The functionalization of 5ʹ-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4ʹ-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5ʹ-DMT-protected ONs could allow producing both symmetric and asymmetric 5ʹ-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5ʹ-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5ʹ-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5ʹ-DMT modification of dsDNA does not affect activity of T5 5ʹ,3ʹ-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5ʹ-DMT-modification or search of ways to separate long 5ʹ-DMT-ssDNA and 5ʹ-OH-ssDNA could allow finding application of 5ʹ-DMT-modified oligo- and polynucleotides.

Mercury Stable Isotope Fractionation during Abiotic Dark Oxidation in the Presence of Thiols and Natural Organic Matter.

Mercury (Hg) stable isotope fractionation has been widely used to trace Hg sources and transformations in the environment, although many important fractionation processes remain unknown. Here, we describe Hg isotope fractionation during the abiotic dark oxidation of dissolved elemental Hg(0) in the presence of thiol compounds and natural humic acid. We observe equilibrium mass-dependent fractionation (MDF) with enrichment of heavier isotopes in the oxidized Hg(II) and a small negative mass-independent fractionation (MIF) owing to nuclear volume effects. The measured enrichment factors for MDF and MIF (ε202Hg and E199Hg) ranged from 1.10‰ to 1.56‰ and from -0.16‰ to -0.18‰, respectively, and agreed well with theoretically predicted values for equilibrium fractionation between Hg(0) and thiol-bound Hg(II). We suggest that the observed equilibrium fractionation was likely controlled by isotope exchange between Hg(0) and Hg(II) following the production of the Hg(II)-thiol complex. However, significantly attenuated isotope fractionation was observed during the initial stage of Hg(0) oxidation by humic acid and attributed to the kinetic isotope effect (KIE). This research provides additional experimental constraints on interpreting Hg isotope signatures with important implications for the use of Hg isotope fractionation as a tracer of the Hg biogeochemical cycle.

English

The properties of xylanase purified from Fusarium heterosporum that was grown in barley-brewing residue under solid-state fermentation and the effects of thiol compounds on the reactivation of the metal ion-inhibited xylanase were investigated. Xylanase was purified to homogeneity by ion exchange chromatography, and its molecular mass was estimated to be 19.5 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the xylanase was 5.0, and it was stable in acidic pH (4.5 to 5.5), where it retained more than 87% of its activity after 24 h. The optimum temperature was 50°C, and it had a half-life of 53 min at 45°C. The apparent Km and Vmax values for the xylanase were 5.63 mg/ml and 800 µmol/mg/min, respectively. Ba2+, Ca2+, Mg2+ and the thiol compounds β-mercaptoethanol and dithiothreitol (DTT) enhanced xylanase activity, while Hg2+, Pb2+ and Zn2+ strongly inhibited enzyme activity. Furthermore, this xylanase had an alternative mode of regulation in the presence of thiol compounds because the enzyme was able to recover its catalytic activity after inhibition by heavy metal ions.   Key words: Hemicellulase, fungus, solid-state fermentation, barley brewing residue, thiol compounds.

Agrobacterium tumefaciens-mediated genetic transformation of sesame (Sesamum indicum L.)

Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. Green shoots recovered from A. tumefaciens-infected explants on selection medium [Murashige and Skoog (MS) basal medium containing 25.0 μM benzyladenine (BA), 25.0 mg l−1 kanamycin and 400.0 mg l−1 cefotaxime] were rooted on MS basal medium containing 2.0 μM indole-3-butyric acid and 5.0 mg l−1 kanamycin. The rooted shoots were established in soil and grown to maturity to collect seeds. The presence, integration and expression of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and GUS histochemical assay, respectively. GUS activity was detected in vegetative and reproductive parts of T0 and T1 plants. Presence of thiol compounds in coculture medium and kanamycin selection at shoot regeneration and at rooting stages were found to be critical for transformation. The transgenes were inherited in Mendelian fashion in T1 progeny as detected by PCR. RT-PCR analysis of T1 plants confirmed the presence of transcripts of uidA gene. The transformation frequency was 1.01%, and 22–24 weeks were required from seed to seed generation time. This protocol can be used to transfer new traits in sesame for quantitative and qualitative improvement.

Regulatory elements and functional implication for the formation of dimeric visinin‐like protein‐1

Size exclusion chromatographic analyses showed that Ca(2+)-free VILIP-1 contained both monomeric and dimeric forms, while no appreciable dimerization was noted with Ca(2+)-free VILIP-3. Swapping of EF-hands 3 and 4 of VILIP-1 with those of VILIP-3 caused the inability of the resulting chimeric protein to form dimeric protein. Nonreducing SDS-PAGE analyses revealed that most of the dimeric VILIP-1 was noncovalently bound together. Reduced glutathione (GSH)/oxidized glutathione (GSSG) treatment notably enhanced the formation of disulfide-linked VILIP-1 dimer, while Ca(2+) and Mg(2+) enhanced disulfide dimerization of VILIP-1 marginally in the presence of thiol compounds. Cys-187 at the C-terminus of VILIP-1 contributed greatly to form S-S-crosslinked dimer as revealed by mutagenesis studies. The ability of GSH/GSSG-treated VILIP-1 to activate guanylyl cyclase B was reduced by substituting Cys-187 with Ala. Together with disulfide dimer of VILIP-1 detected in rat brain extracts, our data may imply the functional contribution of disulfide dimer to the interaction of VILIP-1 with its physiological target(s).

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence, reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.

Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah

Selective reduction on the Cys28–Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28–Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28–Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28–Cys32.

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX–M6PHSA at a sustained slow rate. PTX–M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX–M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX–M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte–cumulus cells functional coupling and intracellular concentration of glutathione

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus–oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3 h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24 h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.

Thermal Polymerization of 1,2-Dithiane

Thermal polymerization of six-membered cyclic disulfide, 1,2-dithiane (DT) was investigated under various polymerization conditions. The polymerization of DT depends strongly on the monomer concentration, and the polymerization did not proceed at the monomer concentration below 4.0 mol/L in feed. In the polymerization of DT in benzene, the polymerization did not effectively proceed even in the presence of typical radcial initiators. From a kinetic study, overall activation energy for bulk polymerization of DT was estimated to be 22.6 kJ/mol. The polymerization of DT was inhibited by an addition of radical inhibitors, and decreased remarkably in the presence of thiol compounds, indicating that the propagation proceeds by a radical intermediate. The molecular weight of the polymers increased with reaction time. Namely, the cyclic polymer is formed mainly by back biting reaction mechanism during reaction, which is related to the formation of polycatenane.

In Vitro Molybdenum Ligation to Molybdopterin Using Purified Components

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme.

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.

Determinação da produção de metalo-beta-lactamases em amostras de Pseudomonas aeruginosa isoladas em João Pessoa, Paraíba

Bactérias produtoras de metalo-beta-lactamases (MBLs) são em grande parte resistentes aos betalactâmicos de largo espectro, incluindo oximino-aminotiazol cefalosporinas e também aos carbapenens. O objetivo deste trabalho foi detectar cepas de Pseudomonas aeruginosa resistentes ao imipenem e à ceftazidima, assim como identificar aquelas produtoras de MBLs. Foram estudadas 198 linhagens não-repetitivas isoladas de diversas amostras clínicas, hospitalares e comunitárias, identificadas bioquimicamente por técnicas de rotina. A triagem para a detecção de amostras produtoras de MBLs foi realizada pelo método de dupla difusão proposto por Arakawa et al. (2000) e modificado por Nakajima et al. (2001), utilizando discos contendo mercaptoacetato de sódio. Foi detectado um percentual de resistência de 19,7% (39/198) ao imipenem e de 15,2% (30/198) à ceftazidima, com 10,1% (20/198) de resistência cruzada aos dois antimicrobianos. Entre estas amostras, 2% (4/198) demonstraram produção de MBLs e padrão de multirresistência. A detecção destas amostras configura um problema emergente, com importantes implicações na terapêutica antimicrobiana, necessitando, portanto, de maior investigação através de metodologia molecular, para melhor caracterizar a extensão do problema.

Inhibition of Bovine Kidney Low Molecular Mass Phosphotyrosine Protein Phosphatase by Uric Acid

Uric acid inhibited 50% of the activity of bovine kidney low molecular mass phosphotyrosine protein phosphatase at concentrations of 1.0, 0.4, 1.3, and 0.2 mM, respectively for p -nitrophenyl phosphate (p -NPP), flavine mononucleotide, β -naphthyl phosphate and tyrosine phosphate (Tyr-P) as substrates. The mixed type inhibition of p -NPP hydrolysis was fully reversible, with K ic and K iu values of 0.4 and 1.1 mM, respectively; the inhibition by uric acid shifted the pH optimum from 5.0 to 6.5. When Tyr-P was the substrate, competitive inhibition was observed with a K i value of 0.05 mM. Inhibition studies by uric acid in the presence of thiol compounds, and preincubation studies in the presence of inorganic phosphate suggest that the interaction of uric acid with the enzyme occurred at the active site, but did not involve SH residues, and that the mechanism of inhibition depended on the structure of the substrates.

Control of polymer structure by a chain-transfer reaction in the radical polymerization of acrylamide by β-mercaptopropionic acid and 1,2,6-hexanetriol trithioglycolate

Star polymer synthesis by radical polymerization of acrylamide (AM) in the presence of thiol compounds was investigated. When 1,2,6-hexanetriol trithioglycolate (HTTG), as a trithiol compound, was used the molecular weights of the polymers were about 3 times those obtained in the presence of β-mercaptopropionic acid, as a monothiol compound, at the same concentration of thiol compounds in the feed. From viscosity measurements of the polymers and analysis of the 1H NMR spectra of the polymers, we presumed that a star-shaped polymer of AM could be synthesized by radical polymerization in the presence of HTTG.

Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio)

Natural killer enhancing factor (NKEF) is a member of the newly defined peroxiredoxin (Prx) family. Its functions are to enhance the cytotoxic capacity of natural killer cells and to prevent DNA and protein from being damaged by oxidative stress in the presence of thiol compounds. However, little is known about the structure and function of NKEF in lower vertebrates. We have recently cloned a cDNA encoding NKEF from the common carp (Cyprinus carpio) by use of suppression subtractive hybridization (SSH). In the present study, we used PCR to obtain a genomic DNA which covers the entire coding region of carp NKEF. In the 3363bp-long genomic sequence, six exons and five introns were identified. The carp NKEF gene has splice donor/acceptor site sequences at the boundaries of exons and introns, and contains two Val-Cys-Pro (VCP) motifs. The exon/intron organization of the carp NKEF gene shows complete conservation with other members of the Prx family. Genomic Southern blotting analyses suggest that carp has multiple copies of the NKEF gene. RT-PCR analyses reveal that carp NKEF has very different expression levels not only in tissues but also from individuals.

Effect of Lysine, Tyrosine, Cysteine, and Glutathione on the Oxidative Cross-Linking of Feruloylated Arabinoxylans by a Fungal Laccase

The potential of a laccase from the fungus Pycnoporus cinnabarinus MIC11 to link covalently some amino acids (tyrosine, lysine, cysteine, and oxidized and reduced glutathione) to the ferulic acid esterified in wheat arabinoxylans was investigated, using capillary viscometry, SE-HPLC, and RP-HPLC of phenolic and thiol compounds. The laccase was compared to the system hydrogen peroxide/horseradish peroxidase. Both oxidative systems were able to gel arabinoxylan solutions by coupling feruloyl esters of adjacent chains into dehydrodimers. Cysteine and reduced glutathione hindered gelation, whereas tyrosine, lysine, and oxidized glutathione had no effect. Under the experimental conditions, in the presence of thiol compounds, a time delay proportional to the thiol concentration was observed. During this period, no esterified ferulic acid was consumed. Cysteine was not directly oxidized either by free ferulic acid or by laccase. When free ferulic acid, cysteine, and laccase reacted together, cysteine was readily...