Amperometric Detection of Catechol and Catecholamines by Immobilized Laccase from DeniLite

DeniLite laccase immobilized Pt electrode was used for detection of catechol and catecholamines. The enzymatically oxidized substrates were measured amperometrically. The sensitivities are 210, 75, 60 and 45 nA/μM with the upper limits of linear ranges of 58, 40, 55 and 55 μM and the detection limits (S/N=3) of 0.07, 0.2, 0.3 and 0.4 μM for catechol, dopamine (DA), norepinephrine (NEPI) and epinephrine (EPI), respectively. The response time (t_90%) is about 2 seconds for each substrate and the long‐term stability is around 40–50 days with retaining 80% of initial activity. The very fast response and the remarkable long‐term stability are the principal advantages of this sensor. In case of catechol, the pH response of the sensor is mainly determined by enzyme's pH profile, however, in case of catecholamines, both enzyme's pH profile and reversibility of the substrate are operated and the optimal pHs for NEPI and EPI shift towards acidic range compared to that for DA. The presence of ascorbic acid (<50 μM) did not interfere with the measurement.     

Laccase-catalyzed carbon-carbon bond formation: oxidative dimerization of salicylic esters by air in aqueous solution

The laccase catalyzed oxidative dimerization of salicylic esters, a rare example of a laccase-catalyzed carbon-carbon bond formation, was studied. This reaction allows the use of air as stoichiometric oxidant and proceeds in aqueous solution. The preparative scope and the mechanism of the method, which provides a new and convenient access to functionalized biaryls under mild conditions, were investigated.     

Laccase Biosensor on Monolayer‐Modified Gold Electrode

Laccase enzymes from two different sources, namely, tree laccase from Rhus vernicifera and fungal laccase from Coriolus hirsutus were used for the development of biosensor for catechol. Laccase was immobilized onto the amine terminated thiol monolayers on gold surface by glutaraldehyde coupling. From the different thiol monolayers investigated, cystamine was found to be optimal with respect to sensitivity, stability, reproducibility, and other electrochemical properties of the enzyme electrode. Linear calibration in the range between 1 and 400 μM for catechol was obtained for fungal laccase covalently coupled on the electrode surface. The kinetic parameters determined using the Lineweaver‐Burk and Eadie‐Hofstee plots were K_m=0.65 mM and V_max=24.5 μA for fungal laccase compared to K_m=5.4 mM and V_max=6.6 μA for tree laccase on cystamine monolayer. The electrode showed good stability for 1 month without loosing appreciable activity when stored dry in a refrigerator at ?20 °C.     

线粒体和亚线粒体体外代谢及漆酶影响的微量热研究

首次将微量热用于亚细胞层次的研究，测定了鲤鱼肝脏线粒体和亚线粒体代谢的热谱及其漆树漆酶的影响，并用ＭＴＤ方程和演化计算技术对它们的代谢热动力学参数进行了全局优化。     

Development of a laccase-based flow injection electrochemical biosensor for the determination of phenolic compounds and its application for monitoring remediation of Kraft E1 paper mill effluent

This paper describes the development of a new system for amperometric determination of phenolic compounds, and its application for monitoring these compounds in paper mill effluent. The method was based on a flow system, a dialysis sampler, and a laccase-based biosensor. The performance of this system was investigated with respect to pH, ionic strength, working potential, and flow-rate dependence. The biosensor showed an excellent long-term stability allowing measurements for over than 3 months. The sensitivity of laccase-based biosensor was tested for phenol, p-chlorophenol, guaiacol and chloroguaiacol; the detector presented selective measurements of micromolar concentration of these compounds. The integration of a dialysis membrane sampling in the system protected the biosensor surface from fouling and gave independence of sample conditions that commonly influence the biosensor performance. These favorable characteristics allowed its application for direct measurements in complex media with no sample pretreatment. This ability was confirmed employing this system in a continuous analysis of phenolic compounds during the remediation of paper mill effluent by ozonization process.     

A Comparative Study of Biocathodes Based on Multiwall Carbon Nanotube Buckypapers Modified with Three Different Multicopper Oxidases

14 Single‐ and multi‐walled carbon nanotubes from different sources were characterized in detail, and the characteristics obtained were carefully analyzed. The carbon material with the highest capacitance, and also other superior properties (“Taunit‐M” from “NanoTechCenter”, Russia), was chosen for further modification and fabrication of buckypaper based electrodes. These electrodes were biomodified with plant and fungal laccases, as well as fungal bilirubin oxidase. The designed biocathodes were investigated in simple buffers and also in a complex physiological fluid (human serum). Biocathodes based on immobilized fungal laccase were bioelectrocatalytically inactive in chloride containing media at neutral pH. In spite of the quite high current densities realized using biodevices based on plant laccase and fungal bilirubin oxidase, the limited thermal stability of the enzymes renders the biocathodes inadequate for practical applications in implanted situations.     

Development of Biosensor for Catechol Using Electrosynthesized Poly(3‐methylthiophene) and Incorporation of LAC Simultaneously

Simultaneous electropolymerization of 3‐methylthiophene and incorporation of Laccase (LAC) was carried out in the presence of propylene carbonate as a medium by amperometric method. This enzyme modified electrode was used for the sensing of polyphenol. Catechol is taken as a model compound for the study. UV‐Vis spectral studies suggest no denaturation of LAC in presence of propylene carbonate. The SEM studies reveal the surface morphology and incorporation of LAC in P3MT with agglomerated flaky masses are observed in with and without enzyme micrographs. The cyclic voltammograms were recorded for 0.01 mM catechol on plain glassy carbon, polymer and enzyme incorporated electrodes at pH 6.0 and scan rate 50 mV s^?1. The fabricated electrochemical biosensor was used for the determination of catechol in aqueous solution by Differential Pulse Voltammetry (DPV) technique. The concentration linear range of 8×10^?8 to 1.4×10^?5 M a value of Michealis? Menten constant K_m=7.67 µmol dm^?3 and activation energy is 32.75 kJ mol^?1. It retains 83 % of the original activity after 60 days which is much higher than that of other biosensors. The developed biosensor was used to quantify catechol in the determination in real samples.     

石榴状凝胶微球固定化漆酶的制备、 表征及降解双酚A

采用铜/锌复合金属磷酸盐晶体和海藻酸钙凝胶双重包覆技术对漆酶进行固定化, 制得石榴状Alg@Cu₃/Zn₃(PO₄)₂@Lac的凝胶微球. SEM, EDX和FTIR表征结果表明, 在凝胶微球内部, 漆酶被成功固定于由海藻酸钙凝胶包覆的铜/锌复合金属磷酸盐晶体内, 铜/锌复合金属磷酸盐晶体镶嵌于海藻酸钙凝胶网格的孔隙中而呈石榴状. 以2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)为底物, 经酶学性质研究表明, 在无机盐晶体和海藻酸钙凝胶的双重保护下, Alg@Cu₃/Zn₃(PO₄)₂@Lac的耐热性、 耐酸性以及储存稳定性比游离漆酶均有不同程度增强. 将Alg@Cu₃/Zn₃(PO₄)₂@Lac应用于双酚A(BPA)的降解, 采用孔径约1 mm滤网实现快速回收, 经6次循环利用, 对BPA的降解率下降约14%, 显示出比较稳定的重复利用性和便捷的可操作性, 这主要得益于海藻酸钙和铜/锌无机盐晶体对漆酶蛋白分子的双重保护.     

Influence of interfacial properties on Ostwald ripening in crosslinked multilayered oil-in-water emulsions

The influence of interfacial crosslinking, layer thickness and layer density on the kinetics of Ostwald ripening in multilayered emulsions at different temperatures was investigated. Growth rates of droplets were measured by monitoring changes in the droplet size distributions of 0.5% (w/w) n-octane, n-decane, and n-dodecane oil-in-water emulsions using static light scattering. Lifshitz-Slyozov-Wagner theory was used to calculate Ostwald ripening rates. A sequential two step process, based on electrostatic deposition of sugar beet pectin onto fish gelatin or whey protein isolate (WPI) interfacial membranes, was used to manipulate the interfacial properties of the oil droplets. Laccase was added to the fish gelatin-beet pectin emulsions to promote crosslinking of adsorbed pectin molecules via ferulic acid groups, whereas heat was induced to promote crosslinking of WPI and helix coil transitions of fish gelatin. Ripening rates of single-layered, double-layered and crosslinked emulsions increased as the chain length of the n-alkanes decreased. Emulsions containing crosslinked fish gelatin-beet pectin coated droplets had lower droplet growth rates (3.1 ± 0.3 × 10⁻²⁶ m³/s) than fish gelatin-stabilized droplets (7.3 ± 0.2 × 10⁻²⁶ m³/s), which was attributed to the formation of a protective network. Results suggest that physical or enzymatic biopolymer-crosslinking of interfaces may reduce the molecular transport of alkanes between the droplets in the continuous phase.     

Green and mild laccase-catalyzed aerobic oxidative coupling of benzenediol derivatives with various sodium benzenesulfinates

The oxidative coupling reaction between hydroquinone or catechols and various sodium benzenesulfinates was investigated using the laccase from Trametes versicolor, in the presence of O₂ in a phosphate buffer solution at room temperature to afford sulfonyl benzenediols in 75–95% yield.