2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and veratryl-alcohol

Environmental concerns have brought attention to the requirement for more efficient and renewable processes for chemicals production. Lignin is the second most abundant natural polymer, and might serve as a sustainable resource for manufacturing fuels and aromatic derivatives for the chemicals industry after being depolymerised. In this work, the mediator 2,2'-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) diammonium salt (ABTS), commonly used with enzyme degradation systems, has been evaluated by means of cyclic voltammetry (CV) for enhancing the oxidation of the non-phenolic lignin model compound veratryl alcohol and three types of lignin (organosolv, Kraft and lignosulfonate) in the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate, ([C2mim][C2SO4]). The presence of either veratryl alcohol or organosolv lignin increased the second oxidation peak of ABTS under select conditions, indicating the ABTS-mediated oxidation of these molecules at high potentials in [C2mim][C2SO4]. Furthermore, CV was applied as a quick and efficient way to explore the impact of water in the ABTS-mediated oxidation of both organosolv and lignosulfonate lignin. Higher catalytic efficiencies of ABTS were observed for lignosulfonate solutions either in sodium acetate buffer or when [C2mim][C2SO4] (15 v/v%) was present in the buffer solution, whilst there was no change found in the catalytic efficiency of ABTS in [C2mim][C2SO4]-lignosulfonate mixtures relative to ABTS alone. In contrast, organosolv showed an initial increase in oxidation, followed by a significant decrease on increasing the water content of a [C2mim][C2SO4] solution.

Topics: Benzothiazoles; Benzyl Alcohols; Biomass; Buffers; Catalysis; Efficiency; Electrochemistry; Energy Transfer; Imidazoles; Indicators and Reagents; Ionic Liquids; Lignin; Oxidation-Reduction; Sulfonic Acids; Viscosity; Water

Coptotermes formosanus Shiraki is a well-known wood-feeding termite, which can degrade not only cellulose and hemicellulose polysaccharides, but also some aromatic lignin polymers with its enzyme complex to the woody biomass. In this study, a very abundant protein was discovered and purified, using a three-step column chromatography procedure, from the tissue homogenate of the salivary glands and the gut of C. formosanus. Mass spectrometric analysis and the following peptide searching against the mRNA database toward this termite species indicated that the novel protein was a hemocyanin enzyme, termed as Hemo1, which further exhibited a strong oxidase activity in the substrate bioassays toward ABTS [2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], as well as other aromatic analogues, such as catechol and veratryl alcohols. This oxidative protein was an acid-favored enzyme with a molecular weight at 82 kDa, and highly active at 80 °C. These findings indicated that the novel protein, hemocyanin, discovered from the gut system of C. formosanus, might be an important ligninolytic enzyme involved in the biomass pretreatment processing, which will potentially enhance the digestibility and utilization of biomass polysaccharides in termite digestive systems.

Topics: Amino Acid Sequence; Animals; Benzothiazoles; Benzyl Alcohols; Catechols; Enzyme Stability; Gastrointestinal Tract; Hemocyanins; Hot Temperature; Insect Proteins; Isoptera; Kinetics; Lignin; Molecular Sequence Data; Molecular Weight; Oxidoreductases; Peptide Mapping; Salivary Glands; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Sulfonic Acids; Wood

Sixteen phenolic compounds, 14 of which naturally occurring, were compared to the synthetic 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and violuric acid (VA) in terms of their ability to act as mediators/enhancers in: (1) laccase oxidation of veratryl alcohol as a lignin model compound, and (2) electrochemical oxidation of kraft and flax lignins. HPLC analysis revealed that the syringyl-type phenols methyl syringate and acetosyringone were the most efficient natural enhancers in the laccase oxidation of veratryl alcohol. Both compounds, though far from the performance of ABTS were able to generate veratraldehyde in amount similar to that obtained with VA. By contrast, the best performing phenolic enhancers for the electrochemical oxidation of lignins were sinapinaldehyde, vanillin, acetovanillone, and syringic acid. Catalytic efficiencies close to those achieved with ABTS and VA were calculated for these phenolic compounds.

Topics: Acetophenones; Benzothiazoles; Benzyl Alcohols; Chromatography, High Pressure Liquid; Electrochemical Techniques; Gallic Acid; Laccase; Lignin; Oxidation-Reduction; Phenols; Sulfonic Acids

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.

Topics: Benzothiazoles; Benzyl Alcohols; Culture Media; Eucalyptus; Glycine max; Industry; Laccase; Oxidation-Reduction; Oxidoreductases; Paper; Streptomyces; Substrate Specificity; Sulfonic Acids

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.

Topics: Basidiomycota; Benzothiazoles; Benzyl Alcohols; Chromatography, High Pressure Liquid; Electrochemistry; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Laccase; Molecular Weight; Oxidation-Reduction; Pyrazoles; Spectrophotometry; Substrate Specificity; Sulfonic Acids

The mechanism of oxidation of lignin by laccase and mediator has been investigated by cyclic voltammetry and bulk electrolysis. Electrochemical properties and reactivities of the two mediators 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT) were studied and their intermediates responsible for lignin oxidation were characterized. ABTS was oxidized first to the cation radical (ABTS+.) and then to the dication (ABTS2+). The two oxidized species are relatively stable and electrochemically reversible, with formal redox potentials (E0' vs Ag/AgCl) of 472 mV for ABTS/ABTS+. and 885 mV for ABTS+./ABTS2+. The dication was shown to be the intermediate responsible for the oxidation of the non-phenolic lignin model compound veratryl alcohol, whereas the cation radical reacted only with phenolic structures in lignin. Cyclic voltammetry of HBT shows only one oxidation peak at 878 mV, but unlike ABTS, the oxidized intermediate was not stable and decayed rapidly. The radical intermediate of HBT was shown to catalyze the oxidation of veratryl alcohol to veratraldehyde. The kinetics of homogeneous redox catalysis of mediators and veratryl alcohol were estimated. ABTS-mediated lignin oxidation at the redox potential of laccase (585 mV) was shown to be possible, but at a very slow rate, as previously reported for laccase and mediator.

Topics: Benzothiazoles; Benzyl Alcohols; Catalysis; Electrochemistry; Indicators and Reagents; Laccase; Lignin; Models, Chemical; Oxidation-Reduction; Oxidoreductases; Sulfonic Acids; Triazoles

Yellow and blue forms of laccase from solid-state and submerged cultures of Panus tigrinus were isolated. Both laccases had similar molecular masses and specific activity, but yellow laccase had no 'blue' maximum in the absorption spectrum. Blue laccase oxidized veratryl alcohol and a nonphenolic dimeric lignin model compound only in the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as electron-transfer mediator. Yellow laccase catalyzed these reactions without any additional compounds. It is supposed that yellow laccase is formed as a result of blue laccase modification by products of lignin degradation. These compounds might play a role of natural electron-transfer mediators for the oxidation of non-phenolic substances, catalyzed by yellow laccase.

Topics: Basidiomycota; Benzothiazoles; Benzyl Alcohols; Electron Transport; Laccase; Lignin; Oxidation-Reduction; Oxidoreductases; Spectrophotometry; Substrate Specificity; Sulfonic Acids