Novel laccase redox mediators

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4-methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3′-sulfophenyl)-3-methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3′-Sulfophenyl)-3-methylpyrazolone-5 was about 30–40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commerical interest for oxidoreductase-catalyzed biodegradation of organic compounds.     

Catalysis of novel enzymatic iodide oxidation by fungal laccase

A fungal laccase (Myceliophthom thermophila) has been shown to function as an iodide oxidase. Unlike other halides which interact with the type 2 copper site and are inhibitors for the laccase, iodide interacts with the type 1 copper site and serves as a substrate capable of donating an electron to the laccase. Under anaerobic conditions, the interaction between the laccase and iodide results in the reduction of the laccase type 1 copper and the concomitant oxidation of iodide to form iodide. In aerated solutions, the laccase catalyzes the oxidation of iodide to iodine and the concomitant reduction of dioxygen to water. The reaction exhibits typical Michaelis kinetics with aK _m of 0.16 ± 0.02M and ak _cat of 2.7 ± 0.2 turnovers per min at the optimal pH (3.4). The catalysis can be enhanced by 2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid), which shuttles electrons rapidly between iodide and the laccase. Bilirubin oxidase also demonstrates significant iodide oxidase activity, suggesting that the property could be a common feature for copper-containing oxidases. Possible industrial and medicinal applications for a laccase-based iodine production system are discussed.     

Syntheses of aromatic aldehydes by laccase without the help of mediators

This communication reports the selective bioconversions of substituted toluenes to substituted benzaldehydes without the help of any mediators by purified laccase of indigenous fungal strain Fomes durissimus microbial type culture collection (MTCC)-1173. Molecular mass of laccase purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to be 74.86 kDa (～75 kDa). By using this purified laccase, selective bioconversions of 3-nitrotoluene to 3-nitrobenzaldehyde, 2-fluorotoluene to 2-fluorobenzaldehyde, 4-fluorotoluene to 4-fluorobenzaldehyde, 2-chlorotoluene to 2-chlorobenzaldehyde and 4-chlorotoluene to 4-chlorobenzaldehyde have been done without the help of mediator molecules within 1–2 hrs at room temperature and pressure with high yields (>90%). All the above bioconversions are good examples of green chemistry.     

Novel laccase redox mediators: spectral, electrochemical, and kinetic properties

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds.     

Comparing the catalytic efficiency of ring substituted 1-hydroxybenzotriazoles as laccase mediators

A series of ring substituted 1-hydroxybenzotriazoles (6-X-HBTs) have been tested as mediators in the laccase-promoted oxidation of 4-methoxybenzyl alcohol, 3,4-dimethoxybenzyl alcohol, and the dimeric lignin model 1-(3,4-dimethoxyphenyl)-2-phenoxyethanol. The effect of the aryl substituents on the yields of oxidation products is remarkable. The catalytic mediation efficiency increases as the electron releasing (ER) properties of the substituent increases up to a maximum value for 6-CH₃-HBT, which resulted a very efficient mediator. Both the oxidation of the 6-X-HBTs to the N-oxyl radicals (6-X-BTNO) by laccase and the hydrogen atom transfer (HAT) process from the benzylic C–H to the 6-X-BTNO contribute to the overall reactivity. The former process is favored by ER substituents that lower the mediator redox potentials. On the other hand, ER substituents decrease the 6-X-BTNO reactivity in the HAT process due to a decrease in the NO–H BDE value, as assessed in this study through a radical equilibration technique.     

Comparison of TEMPO and its derivatives as mediators in laccase catalysed oxidation of alcohols

A series of TEMPO (2,2′,6,6′-tetramethylpiperidinyl-1-oxy) derivatives were studied as mediators of laccase (from Trametes versicolor) in the oxidation of benzyl alcohol and 1-phenylethyl alcohol. TEMPO (1), 4-hydroxy-TEMPO (2) and 4-acetylamino-TEMPO (4) turned out to be the most active mediators for laccase. In addition, 4-acetylamino-TEMPO and 4-hydroxy-TEMPO were more active in the oxidation of 1-phenylethanol compared to TEMPO. For these mediators kinetic isotope effects in the range of 2.1-3.2 were observed for α-monodeutero-p-methylbenzyl alcohol oxidation. These values are consistent with a mechanism involving oxoammonium intermediacy. Competition experiments between benzyl alcohol and 1-phenylethanol showed that TEMPO and its derivatives react faster with primary alcohols than with secondary alcohols, also in line with the proposed mechanism.     

Novel beta-lactam antibiotics synthesized by amination of catechols using fungal laccase

Novel cephalosporins, penicillins, and carbacephems were synthesized by amination of catechols with amino-beta-lactams like cefadroxil, amoxicillin, ampicillin and the structurally related carbacephem loracarbef using laccase from Trametes sp. All isolated monoaminated products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant Enterococci. Observed differences in the cytotoxicity and in vivo activity in a "Staphylococcus-infected, immune suppressed mouse" model are discussed.     

In vitro evolution of a fungal laccase in high concentrations of organic cosolvents

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase--R2 variant--was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic copper varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.     

Electrochemistry and oxygenation kinetics of tetradentate Schiff base manganese(II) complexes

The electrochemistry and oxygenation kinetics of a series of Mn(II) complexes containing ligands derived from substituted salicylaldehyde and linear diamines have been studied. Complexes prepared from ethylenediamine produce cyclic voltammograms with a quasi-reversible Mn(II)-Mn(III) couple and oxygenation reactions which are first-order in both complex and O₂, indicating formation of an Mn(III)-O⁻₂ species as the slow step in the reaction. The case of oxidation/oxygenation increases with increasing electron-donating ability of substituents on the salicyladehyde rings. Complexes with from six to 10 methylene groups in the diamine precursor produce highly irreversible voltammograms and oxygenation data which can not be interpreted in terms of a simple mechanism. Polymerization of the complexes is advanced as an explanation for these effects.     

Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds

Fungal laccase was immobilized on carbon-fiber electrodes using classical methods: physical adsorption, glutaraldehyde, carbodiimide and carbodiimide/glutaraldehyde. The highest biosensor response was obtained using carbodiimide/glutaraldehyde for coupling laccase to carboxyl groups on the carbon fibers. In this method, different percentages of glutaraldehyde had important effects on the sensitivity of the biosensor, the best percentage of glutaraldehyde being 10% (m/v). The behavior of the obtained biosensor was investigated in terms of sensitivity, operational range, pH and applied potential. The developed biosensor showed an optimum response at pH 5.0 and at an applied potential of -100 mV. The immobilized laccase retained a good activity for over 2 months.     

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

Summary An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na₂S₂O₅ inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H₂O₂ as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10^-5 mol dm^-3 and W_max = 8.02 x10^-7 mol dm^-3 sec^-1).     

A spectroscopic investigation of the interaction between nitrogen monoxide and copper sites of the fungal laccase from Rigidoporus lignosus

The interaction of NO, with the copper centres of the laccase secreted by Rigidoporus lignosus was studied under both aerobic or anaerobic conditions. The reduction of the T1 site was always observed, as detected by the disappearance of the characteristic optical band at 604 nm (T3 presents probably the same behaviour because of the decreasing of the band at 330 nm) and the absence of its characteristic EPR signal, while T2 undergoes an initial partial and transitory reduction, its EPR signal intensity totally restoring after 24 h interaction. Different magnetic parameters of the T2 site have been detected, evidencing an increase of the hyperfine coupling constant. Furthermore, the number of superhyperfine lines on the fourth line of T2 copper was also found to increase from seven in the native to nine in the NO-treated laccase, this fact implying the coordination of a nitrogenous species to the T2 site. It was also shown that nitrite can be a source of NO, thus, paralleling the behaviour of NO-donor molecules or NO gas, but after longer interaction times. The nitrogenous species coordinated to T2 site is probably NO2-, which arises indirectly by NO oxidation. In order to understand the mechanistic pathway of this interaction, some experiments were also carried out in the presence of azide to study the interaction of NO with this laccase having its trinuclear cluster blocked by the presence of an exogenous ligand as N3-. After the addition of NO-donor molecules to the azide-treated laccase, a new EPR signal appeared at low temperatures, which is ascribable to the partially reduced T3 site, while the T1 and T2 sites were found to be totally reduced. The mechanistic pathway of the NO interaction seems to proceed through the reduction of T1 and T3 copper sites, followed by the coordination of nitrogenous species to T2.     

Electrochemistry and homogeneous self-exchange kinetics of the aqueous 12-tungstoaluminate(5-/6-) couple

The effect of alkali metal (M) chloride or triflate supporting electrolytes (0.1-1.0 mol L(-1)) on the midpoint potential E(m) of the aqueous AlW12O40(5-/6-) couple in cyclic voltammetry, after correction (E(corr)) for liquid junction potentials, can be represented in terms of ionic strength according to the extended Debye-Hückel equation. However, unrealistically short AlW12O40(5-/6-)-cation closest-approach distances are required to accommodate the specific effects of M+, and the infinite-dilution potential E(corr)(0) values are not quite consistent from one M+ to another. The pressure dependence of Em is qualitatively consistent with expectations based on the Born-Drude-Nernst theory. The strong accelerating effects of supporting electrolytes on the standard electrode reaction rate constant k(el) at pH 3 as measured by alternating current voltammetry (ACV), and on the homogeneous self-exchange rate constant k(ex) at pH 3-7 as measured by 27Al line broadening, depend specifically on the identity and concentration of M+ (Li+ < Na+ < K+ < Rb+) rather than on the ionic strength, whereas the effect of the nature of the supporting anion (Cl- or CF3SO3-) is negligible. Extrapolation of k(el) and k(ex) to zero [M+] indicates that the uncatalyzed electron transfer rate is negligibly small relative to the M+ catalyzed rates. The kinetic effects of M+ show no evidence of the saturation expected had they been due primarily to ion pairing with AlW12O40(5-/6-). The catalytic effect of M+ operates primarily through lowering the enthalpy of activation, which is partially offset by a strongly negative entropy of activation and, for the homogeneous exchange catalyzed by K+ or Rb+, becomes mildly negative; thus, the catalytic effect of M(+) is enthalpy-driven but entropy-limited. For the electrode reaction, the volume of activation averages +4.5 +/- 0.2 cm(3) mol(-1) for all M+ and [M+], in contrast to the negative value predicted theoretically for the uncatalyzed reaction. These results are consistent with a reaction mechanism, previously proposed for other anion-anion electron-transfer reactions, in which anion-anion electron transfer is facilitated by partially dehydrated M+.     

Electrochemistry and perovskite photovoltaics

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Electrochemistry of peptides

This opinion is focused on the electrochemistry of peptides utilised in biological and technological processes. The redox behaviour of peptides important in biomedicine, neuropeptides and antimicrobial peptides is highlighted. In addition to peptides composed of essential amino acids, peptide-mimetic molecules such as peptide nucleic acid and artificial peptidic wires transferring protons and electrons are reviewed. The application of electrochemistry for the research of peptide–membrane and peptide–surface interactions is also discussed.     

Electrochemistry of nanobubbles

Gas bubble formation is a common phenomenon in numerous electrochemical processes, such as water splitting, chloralkaline process, and fuel cells. Many efforts have been made to understand the behaviors of microsized or larger gas bubbles in electrochemical systems in the past few decades. It was not until recent years that the electrochemistry of nanosized gas bubbles (nanobubbles) has begun receiving attention. In this short review, we summarize recent advances in the field of electrochemistry of nanobubbles, ranging from new fundamental understandings of nanobubble behaviors to the development of novel bubble-based applications inspired by the basic research of nanobubble electrochemistry.