Atomic-scale investigation of the interaction between coniferyl alcohol and laccase for lignin degradation using molecular dynamics simulations and spectroscopy

The interaction between coniferyl alcohol (CA) and laccase (LAC) was investigated using molecular dynamics (MD) simulations and spectral experiments. The mode of interaction between CA and LAC was established by MD simulations. The micro-environmental changes, stability and rigidity of the LAC-CA system were assessed by relevant parameters. These parameters include root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (Rg). The calculated binding free energy (ΔG_binding=??19.99?kcal·mol.^?1), the van der waals (VDW) contribution (ΔG_vdw=?23.99?kcal·mol^?1) and the electrostatic energy (ΔG_ele=?23.09?kcal·mol^?1) of LAC-CA system demonstrated that the interaction of LAC-CA was a spontaneous process and the main interaction forces were van der Waal's and electrostatic forces. The values of ΔG_vdw and ΔG_ele were negative, which demonstrated that VDW interactions and electrostatic interactions were favorable for the binding of CA and LAC. The binding constants, thermodynamic parameters, molecular force types and binding distances confirmed the interaction between CA and LAC and further verified the rationality of the theoretical model by spectral experiments. The MD simulations and experimental approaches provide clues for the discovery of new mediators and useful references for the mechanism of microbial degradation of lignin and industrialization of lignocellulose.     

Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations

The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu^II/I reduction potential (E°′_T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°′_T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°′_T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°′_T1Cu. While minimal and small E°′_T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°′_T1Cu increase. Moreover, the influence of these mutations on E°′_T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°′_T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k_cat/K_M) relative to WT-SLAC.     

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.     

Single‐Enzyme Biofuel Cells

Herein, we demonstrate that the intramolecular electron transfer within a single enzyme molecule is an important alternative pathway that can be harnessed to generate electricity. By decoupling the redox reactions within a single type of enzyme (for example, Trametes versicolor laccase), we harvested electricity efficiently from unconventional fuels including recalcitrant pollutants (for example, bisphenol A and hydroquinone) in a single‐laccase biofuel cell. The intramolecular electron‐harnessing concept was further demonstrated with other enzymes, including power generation during CO₂ bioconversion to formate catalyzed by formate dehydrogenase from Candida boidinii . The novel single‐enzyme biofuel cell is shown to have potential for utilizing wastewater as a fuel as well as for generating energy while driving bioconversion of chemical feedstock from CO₂.     

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.     

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.     

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.     

Optical fiber biosensor coupled to chromatographic separation for screening of dopamine, norepinephrine and epinephrine in human urine and plasma

An optical fiber biosensor has been developed for the determination of catecholamines (dopamine, norepinephrine and epinephrine) based on the recognition capacity of the enzyme laccase. In this study, a glass tube constituted by a fused silica fiber coated with a film of polystyrene/divinylbenzene resin (PS/DVB) was used for catecholamines separation. Firstly, the analyzer was tested for calibration and its analytical performance for catecholamines detection was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED). The developed analytical device shows a high potential for catecholamines quantification with a detection limit of 2.1, 2.6 and 3.4 pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. The analytical sensitivity, inferred from the slope of the calibration curves established for a range of concentrations between 5 and 125 pg mL⁻¹, was found to be 0.344, 0.252 and 0.140 dB/pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. Furthermore, catecholamines speciation with the PS/DVB fiber was completely achieved in 3 min. The analytical performance of the reported sensor was also evaluated and found adequate for catecholamines determination in human urine and plasma samples.     

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.