Electroreduction of laccase covalently bound to organothiol monolayers on gold electrodes

Cerrena unicolor laccase was immobilized on the gold electrode by covalent bonding to self-assembled monolayers of mercaptoundecanoic or mercaptopropionic acids. STM images of immobilized laccase proved high population of the laccase molecules on the monolayer modified electrode. The SERS experiments in concert with resonance Raman effect confirmed that the structure at the “blue” copper site of the immobilized protein remained intact. The accessibility of individual copper sites for electron exchange with the gold electrode surface was investigated by voltammetry. The electrode behavior of laccase is different in the presence and absence of oxygen, showing that the immobilized enzyme is reactive towards oxygen. Addition of two common mediators improved the electrical connectivity of the enzyme with the electrode, increased the catalytic efficiency of immobilized laccase and switched the onset of catalytic current to the potentials of the mediator. Immobilization of laccase on well-organized mercaptoundecanoic acid separates efficiently the enzyme from the electrode and does not allow easy access of mediators to the surface. Attachment of the enzyme at smaller distance from the electrode by means of significantly shorter spacer molecule—mercaptopropionic acid improved the efficiency of catalytic reduction of oxygen on the monolayer modified electrode.     

Hybrid phenol biosensor based on modified phenoloxidase electrode

Electrolytic deposition has been widely used to immobilize biomacromolecules, and it is always the most important factor to preserve or even increase an activity of the immobilized protein. We report here simple and rather universal method for the highly efficient immobilization of laccase for amperometric biosensing. Laccase from Cerrena unicolor has been successfully immobilized (electrolytic deposition) on the surface of thin, ordered polythiophene films (3-methylthiophene/3-thiopheneacetic acid/N-heptyl-3,6-bis(2-thiophene)carbazole). Two different compounds capable of mediating laccase-catalyzed reactions have been tested by cyclic voltammetry. They exhibited quasi-reversible electrodic behaviour with formal redox potentials ranging from 68 and 918 mV (E⁰vs. SCE). The immersion of the laccase-coated electrode in solution with substrate generated large catalytic currents easily recorded by cyclic voltammetry at low potential scan rates. Considering the fact, that immobilization strategy showed high efficiency, obtained results suggest that method for phenoloxidase immobilization has a great potential of enabling high throughput fabrication of bioelectronics’ devices.     

白腐菌去除造纸废水中木质素的机理及研究进展

阐述了白腐菌对造纸废水中木质素去除的应用现状及研究进展,并对白腐菌产生的木素过氧化物酶、锰过氧化物酶、漆酶对木质素的降解机理进行了分析和探讨。     

Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w/v) was mixed into an oil-in-water emulsion system (4.4% w/w oil, 0.22% w/w whey protein, pH 4.5). Two separate, identically composed, emulsion systems were prepared by different methods of preparation. The emulsions prepared separately and subsequently mixed with SBP (referred as Mix A) produced significantly larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G′) with time, were higher (P < 0.05) than the corresponding laccase catalyzed rates, but the final G′ values were higher for laccase catalyzed gels, regardless of the presence of emulsions or type of emulsion preparation (Mix A or Mix B). For both enzymes, rates of gelation in Mix A were higher (P < 0.05) than in Mix B, and higher stress was needed to break the gels in Mix A than in Mix B at similar enzyme dosage levels. These differences may be related to a lower availability of the feruloyl groups for cross-linking when the SBP was homogenized into the emulsion system during preparation.     

Enhanced transformation of triclosan by laccase in the presence of redox mediators

Triclosan (TCS), an antimicrobial agent, is an emerging and persistent environmental pollutant that is often found as a contaminant in surface waters and sediments; hence, knowledge of its degradability is important. In this study we investigated laccase-mediated TCS transformation and detoxification, using laccase (from the fungus Ganoderma lucidum) in the presence and absence of redox mediators. Transformation products were identified using HPLC, ESI-MS and GC-MS, and transformation mechanisms were proposed. In the absence of redox mediator, 56.5% TCS removal was observed within 24 h, concomitant with formation of new products with molecular weights greater than that of TCS. These products were dimers and trimers of TCS, as confirmed by ESI-MS analysis. Among the various mediators tested, 1-hydroxybenzotriazole (HBT) and syringaldehyde (SYD) significantly enhanced TCS transformation (∼90%). The presence of these mediators resulted in products with lower molecular weights than TCS, including 2,4-dichlorophenol (2,4-DCP; confirmed by GC-MS) and dechlorinated forms of 2,4-DCP. When SYD was used as the mediator, dechlorination resulted in 2-chlorohydroquinone (2-CHQ). Bacterial growth inhibition studies revealed that laccase-mediated transformation of TCS effectively decreased its toxicity, with ultimate conversion to less toxic or nontoxic products. Our results confirmed the involvement of two mechanisms of laccase-catalyzed TCS removal: (i) oligomerization in the absence of redox mediators, and (ii) ether bond cleavage followed by dechlorination in the presence of redox mediators. These results suggest that laccase in combination with natural redox mediator systems may be a useful strategy for the detoxification and elimination of TCS from aqueous systems.     

Enzymatic sensing with laccase-functionalized textile organic biosensors

Herein we present a textile wearable electrochemical transistor by functionalizing a single cotton yarn with semiconducting polymer. The organic electrochemical transistor (OECT), which is low cost and completely integrated e-textile, is decorated by adsorption of the fungal laccase POXA1b, and is used as biosensor for the direct detection of Tyrosine (L-Tyr) without the use of electron mediators. The detection of Tyr in real-case scenario such as human physiological fluids would own a paramount importance in noninvasive analysis of the patient's condition, monitoring and preventing several pathologies. To assess the reaction progression, the redox process is studied by UV–visible absorption with test reference molecule of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS): the results confirmed that the oxidation reaction is driven by the presence of laccase enzyme and direct electron transfer occurred. The modulation of the signal response and the kinetic of the signal is used to detect Tyr molecule in aqueous solution and the role of the enzyme adsorption on the textile is analyzed. A kinetic analysis of the characteristic modulation times of the sensing curves, confirm the sensing properties of the textile device. The textile-based biosensor is demonstrated to monitor human health biomarkers through wearable applications in a non-invasive way, finding potential application in sport, healthcare and working safety.     

Oxidation of Polycyclic Aromatic Hydrocarbons using Partially Purified Laccase from Residual Compost of Agaricus bisporus

Laccase partially purified from residual compost of Agaricus bisporus by an aqueous two‐phase system (Lac ATPS) was used in degrading polycyclic aromatic hydrocarbons: fluorene (Flu), phenanthrene (Phe), anthracene (Ant), benzo[a]pyrene (BaP), and benzo[a]anthracene (BaA). The capacity of the enzyme to oxidize polyaromatic compounds was compared to that of the crude laccase extract (CE). After treatment of 72 h, Lac ATPS and CE were not capable of oxidizing Flu and Phe, while Ant, BaP, and BaA were oxidized, resulting in percentages of oxidation of 11.2 ± 1, 26 ± 2, and 11.7 ± 4 % with CE, respectively. When Lac ATPS was used, the following percentages of oxidation were obtained: 11.4 ± 3 % for Ant, 34 ± 0.1 % for BaP, and 13.6 ± 2 % for BaA. The results reported here demonstrate the potential application of Lac ATPS for the oxidation of polycyclic aromatic hydrocarbons.     

传统生漆技术中的化学

大漆主要是由脂类物质组成。漆酚、虫漆酚和硫醇通过漆酶聚合,产生耐久性非常好的涂料,用于漆器及文物。东方大漆是从亚洲一些国家产的漆树上采集的,这些国家包括中国、日本和韩国(Rhus vernicifera)、越南和中国台湾(Rhus succedanea)、泰国和缅甸(Gluta usitata)。在干燥过程中,除水外,没有有机溶剂挥发。近些年绿色化学及可持续发展化学的新理念被提了出来,并考虑用于漆器工艺。大漆是一种来自漆树的天然材料。由于其自固化体系,天然大漆可称为低溶剂、高固含量型的涂料。因此,对于漆液和漆膜的研究一直没有中断。为了让读者对大漆有一个综合全面的了解,结合我们实验室的工作,本文对近些年漆器研究的进展进行了综述,主要包括成分分析、酶的催化、氧化、改性、合成及漆器技术方面的内容。     

Herbal infusions bioelectrochemical polyphenolic index: Green tea – The gallic acid interference

An amperometric biosensor containing immobilised laccase from Trametes versicolor was used for the quantification of phenolic compounds in herbal infusions and green tea samples, from nine botanical families. The main purpose of this research was to correlate the bioelectrochemical polyphenolic index (BPI) of the samples with the total phenolic content according to the Folin–Ciocalteu spectrophotometric method (TPC), and the total antioxidant activity (TAA) measured by TEAC (Trolox Equivalent Antioxidant Capacity). A strong correlation between BPI, TPC, and TAA was obtained for 10 herbal infusion samples. However, when a green tea sample was taken into account, a decrease in the linear correlation coefficient (r²) from 0.9949 to 0.2599 and 0.5609 to 0.1086 for the relationship between BPI/TPC and BPI/TAA, respectively, was observed. Thus, we could conclude that the green tea’s matrix affects the laccase-based biosensor response. HPLC–DAD analysis showed the presence of gallic acid only in the green tea sample. The results have indicated that gallic acid inhibited the laccase activity regarding the ABTS oxidation in a concentration-dependent manner. The very strong correlation between BPI/TPC obtained for herbal infusions allow us to conclude that the laccase-based biosensor, used in this research, provides a valuable tool to obtain a valid estimation of the classical Folin–Ciocalteu index, in an uncomplicated and fast way.