Application of an enzymatic cascade reaction for the synthesis of the emeraldine salt form of polyaniline

The synthesis of the emeraldine salt form of polyaniline (PANI-ES) from aniline with Aspergillus sp. glucose oxidase (GOD), d-glucose, dissolved O2, and horseradish peroxidase isoenzyme C (HRPC) in the presence of large unilamellar vesicles of AOT (sodium bis-(2-ethylhexyl)sulfosuccinate) as templates at pH = 4.3 and T ~ 25 °C was investigated in a systematic way. In this cascade reaction mixture, the oxidation of aniline is catalyzed by HRPC with H2O2 that is formed in situ as byproduct of the GOD-catalyzed oxidation of d-glucose with O2. Under the elaborated experimental conditions which we considered ideal, the formation of PANI-ES products is evident, as judged by UV/Vis/NIR and EPR measurements. Comparison was made with a reference reaction, which was run under similar conditions with added H2O2 instead of GOD and d-glucose. Although the reference reaction was found to be superior, with the cascade reaction, PANI-ES products can still be obtained with high aniline conversion (> 90%) within 24 h as stable dark green PANI-ES/AOT vesicle dispersion. Our results show that the in situ formation of H2O2 does not prevent the inactivation of HRPC known to occur in the reference reaction. Moreover, the GOD used in the cascade reaction is inactivated as well by polymerization intermediates.

Purification and partial characterization of a thermostable peroxidase isoenzyme from Vigna sp seedlings

A peroxidase isoenzyme (named A6 in a previous study) was purified from radicles of a Vigna species by a combination of gel filtration on Sephadex G-100, heat treatment, CM-cellulose chromatography and DEAE-cellulose chromatography. It has been successfully separated from other anionic isoperoxidases expressed in the same tissue. It has a molecular weight of about 41 kDa and exhibits a great activity toward the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at optimum pH (pH 3 for ABTS, pH 4 for OPD and pH 6 for the others) and toward the reduction of H2O2. Its very acid optimum pH for the oxidation of ABTS is not a characteristic of other peroxidases except African oil palm tree peroxidase. Apparent Km values for these substrates were respectively 3.50 mM, 0.12 mM, 1.81 mM, 0.05 mM, 17.22 mM and 2.53 mM; catalytic efficiencies were 5.12×104 mM-1.min-1, 2.22×106 mM-1.min-1, 1.59×105 mM-1.min-1, 1.82×105 mM-1.min-1, 3.17×105 mM-1.min-1and 1.79×106 mM-1.min-1. It has an optimum temperature of activity around 60°C, and its heat inactivation fit to the first-order kinetics, with half-lives of 3.06 weeks, 13.5 hours, 15 min and 3.5 min at 50°C, 70°C, 80°C and 90°C respectively. The calculated activation energy (E) for its thermal inactivation was found to be 221.5 KJ/mol at pH8. This peroxidase isoenzyme is stable for 4 months at room temperature, loosing only 5% of its initial activity over this period. Mg2+ inhibits the activity of the enzyme. The Ca2+ ions greatly increase the stability of this peroxidase at 80°C, while Mn2+ and Zn2+ reduce it. The enzyme is inhibited by sodium azide at concentrations above 1 µM with an IC50 value around 10 µM. This inhibition, in addition to the RZ value (A403nm/A280nm) evaluated at 2.4 confirms the presence at the active site of the enzyme of a heme group common to class III peroxidases. The unusual catalytic and thermal characteristics of A6 could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.

Effect of template type on the preparation of the emeraldine salt form of polyaniline (PANI-ES) with horseradish peroxidase isoenzyme C (HRPC) and hydrogen peroxide

Different types of templates consisting of sulfonate or sulfate groups were compared for the horseradish peroxidase/H2O2-catalysed synthesis of the emeraldine salt form of polyaniline from aniline at pH = 4.3.

Activity and isoenzyme composition of peroxidase in the vegetative organs of Japanese quince under steppe zone conditions

Parameters of peroxidase complex are indicators of the adaptable processes which are taking place in plants under the environment influence. In this work the general activity dynamics and peroxidase isoenzyme composition in vegetative organs of Japanese quince (Chaenomeles Lindl.) were studied. The four species introduced in a steppe zone of Ukraine were researched: Ch. japonica (Thunb.) Lindl., Ch. speciosa (Sweet) Nak., Ch. cathayensis (Hemsl.) Schneid., Ch. × superba (Frahm) Red.). It was determined that the enzyme activity of these species possesses specific features subject to the phases of seasonal development and in response to the action of the hydrothermal stress. The highest enzyme activity in the period of intensive growth and flowering, with a subsequent sharp drop towards the middle of summer and further smoother decrease during the phase of physiological rest were observed in the leaves of Ch. speciosa and Ch. cathayensis. A higher peroxidase activity was a characteristic of Ch. japonica and Ch. × superba in the middle of the vegetative period, which can be considered a display of adaptation processes in the conditions of an unfavourable hydrothermal regime. The dynamics of changes in the quantitative composition of isoperoxidases was similar to the dynamics of enzyme activity. As a result of the generalization of the obtained data it is possible to consider that Ch. japonica and Ch. × superba are the steadiest in the conditions of the region.

Preparation of aqueous polyaniline-vesicle suspensions with class III peroxidases. Comparison between horseradish peroxidase isoenzyme C and soybean peroxidase

Aniline was polymerised enzymatically in aqueous solution at pH = 4.3 and 25°C in the presence of submicrometer-sized vesicles formed from sodium bis(2-ethylhexyl)sulphosuccinate (AOT). H2O2 served as oxidant and the enzyme used was either horseradish peroxidase isoenzyme C (HRPC) or soybean peroxidase (SBP), both being class III peroxidases. From previous studies with HRPC, it is known that stable vesicle suspensions containing the emeraldine salt form of polyaniline (PANI-ES) can be obtained within 1–2 days with a 90–95 % yield, provided that optimal reaction conditions are applied. Unfortunately, HRPC becomes inactivated during polymerisation. In the present study, a linear dendritic block copolymer was added to HRPC, resulting in higher operational enzyme stability; the stabilising effect, however, was too small to afford a substantial decrease in the required amount of enzyme. Moreover, replacing HRPC with SBP was of no advantage, although SBP is known to be more stable towards inactivation by H2O2 than HRPC. By contrast, SBP was found to be much slower in oxidising aniline, and complete inactivation of SBP occurred before all the aniline monomers were oxidised, leading to low yields and the formation of over-oxidised products. The same was observed for HRP isoenzyme A2. Reactions without vesicles indicated that peroxidase inactivation was probably caused by PANI-ES.