Catalysis of novel enzymatic iodide oxidation by fungal laccase

A fungal laccase (Myceliophthom thermophila) has been shown to function as an iodide oxidase. Unlike other halides which interact with the type 2 copper site and are inhibitors for the laccase, iodide interacts with the type 1 copper site and serves as a substrate capable of donating an electron to the laccase. Under anaerobic conditions, the interaction between the laccase and iodide results in the reduction of the laccase type 1 copper and the concomitant oxidation of iodide to form iodide. In aerated solutions, the laccase catalyzes the oxidation of iodide to iodine and the concomitant reduction of dioxygen to water. The reaction exhibits typical Michaelis kinetics with aK _m of 0.16 ± 0.02M and ak _cat of 2.7 ± 0.2 turnovers per min at the optimal pH (3.4). The catalysis can be enhanced by 2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid), which shuttles electrons rapidly between iodide and the laccase. Bilirubin oxidase also demonstrates significant iodide oxidase activity, suggesting that the property could be a common feature for copper-containing oxidases. Possible industrial and medicinal applications for a laccase-based iodine production system are discussed.     

Immobilization of Laccase from Trametes versicolor on Chitosan Macrobeads for Anthracene Degradation

Anthracene bioconversion was achieved by immobilized enzyme technology. An oxidation yield of 0.7?mg/L of polycyclic aromatic hydrocarbons reached 60% following 24?h of incubation with laccase from Trametes versicolor covalently immobilized on glutaraldehyde-activated chitosan at the optimal pH of 5 in the presence of diammonium 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the radical mediator. High-performance liquid chromatography indicated that the main product of anthracene oxidation was 9,10-anthraquinone which is less toxic than its precursor. Highly porous 3?mm diameter chitosan macrobeads were synthesized by precipitation in alkaline solution. Support activation with glutaraldehyde was confirmed by elemental analysis, thermogravimetry, and infrared spectroscopy. The bioreactor system was characterized for kinetic parameters obtaining a Michaelis–Menten constant of 0.13?mM and a maximum rate of 0.0011?µmol/min/mg, thermal stability, and reuse. The protein and glutaraldehyde concentrations were optimized to enhance the efficiency of the bioreactor.     

Implementation of a Simple Nanostructured Bio‐electrode with Immobilized Rhus Vernicifera Laccase for Oxygen Sensing Applications

In this work a simple nanostructured direct‐electron transfer bio‐electrode based on tree laccase from Rhus vernicifera is described. The electrode was implemented on a 2 mm diameter graphite mine casted with a reduced graphene surface presenting the specific capacitance of 195.8 F g⁻¹. About 10 μl of mixture between 25 mg mL⁻¹ laccase suspension and 5 mg mL⁻¹ single‐walled carbon nanotubes in 2 % SDS is dropped over the surface followed by 5 μl of the biological friendly tetrakis(2,3‐dihydroxypropyl)‐silane monomer sol to provide physical entrapment in a silica matrix after gelation. The rigidity of enzyme encapsulation allowed to obtain a constant enzyme turnover of about 16 min⁻¹ in the extended pH range of 6.0‐7.5, being the activity almost proportional to the temperature used in the interval between 25 and 40 °C. The graphite‐graphene/SWCNT‐laccase/sol‐gel electrode enabled a proportional response to molecular oxygen up to the concentration of 0.45 mmol L⁻¹ and is capable to generate the maximum power of 4.5 μW cm⁻² at 0.250 V vs the AgCl/Ag reference electrode in quiescent oxygen saturated solution.     

丁二酸酐修饰对漆酶稳定性和除酚效率的影响(英文)

采用(NH4)2SO4分步沉淀法对诺维信中国公司生产的漆酶制剂DeniLiteIIS进行了纯化,并用丁二酸酐(SA)对纯化酶进行了化学修饰,运用三硝基苯磺酸法、紫外光谱法及荧光光谱法对修饰效果进行了初步表征,比较了天然酶和修饰酶的pH稳定性、热稳定性及除酚效率.结果表明,修饰酶的平均氨基修饰度为85%,其紫外吸收峰和荧光发射峰均出现蓝移,而且紫外吸收减小、荧光强度增加.尽管采用SA化学修饰未能改变漆酶的最适反应温度,但使其最适反应pH值由4.5提高到5.5,并且使酶活提高60%.与天然酶相比,修饰酶的pH稳定性和热稳定性更高,催化效率(kcat)和酶与底物的亲和力(kcat/Km)分别提高了53%和122%,对邻、间和对苯二酚的除酚效率分别提高了48%,57%和18%.这预示着这些修饰漆酶可望应用于工业生产和酚类污染废水的治理.     

高分子聚合物-多壁碳纳米管复合物固定漆酶及其在玻碳电极上的直接电子迁移

采用不同结构的高分子聚合物与纯化的多壁碳纳米管(MWCNTs)共混的方法,制备得到聚合物非共价功能化多壁碳管复合物,测定了这些载体对漆酶(lac)的担载量、固定漆酶的比活力及稳定性.以固定漆酶的复合物修饰玻碳(GC)电极后,采用循环伏安法研究这些电极在无氧磷酸盐缓冲液(PBS)中的直接电化学行为及催化氧还原活力,粗略地测定了固定漆酶与电极间电子转移的速率常数.实验结果表明,当聚合物中含亲漆酶基团或能与漆酶活性中心发生相互作用的官能团时利于直接电子转移,而且复合物固定漆酶保持了游离漆酶的天然构象.这些电极中,lac/NIPAM-co-BPCP-M WCNTs/GC(NIPAM-co-BPCP:N-烯丙基-1-苯甲酰基-3-苯基-4,5-2H-4-甲酰胺基吡唑-co-N-异丙基丙烯酰胺)在无氧PBS中发生直接电子转移的式电位(605mV)更接近漆酶活性中心的式电位(580mV),具有较快的异相电子转移速率(0.726s-1),较高的漆酶担载量(103.5mg/g)和固定漆酶比活力(1.68U/mg),较高的催化氧还原能力(氧还原起始电位820mV,在650mV时的催化峰电流为85.5μA)以及良好的重复使用性和长期使用性.     

Recent developments and applications of screen-printed electrodes in environmental assays—A review

Screen-printed electrodes (SPEs), which are used as economical electrochemical substrates, have gone through significant improvements over the past few decades with respect to both their format and their printing materials. Because of their advantageous material properties, such as disposability, simplicity, and rapid responses, SPEs have been successfully utilised for the rapid in situ analysis of environmental pollutants. This critical review describes the basic fabrication principles, the configuration designs of SPEs and the hybrid analytical techniques based on SPEs. We mainly overview the electrochemical applications of SPEs in environmental analysis over the past 3 years, including the determination of organic compounds, heavy metals and gas pollutants.     

氨基功能化大孔SiO2固定化漆酶

在溶剂热条件下,采用后嫁接法对新型大尺寸Si O2大孔材料进行氨基化改性,以氨基修饰的Si O2大孔材料为载体,借助交联剂戊二醛(GA)使诺维信工业漆酶固定化.研究结果表明:在GA质量分数为3%,p H值为5,漆酶质量浓度为30 mg·m L-1条件下固定化4 h制备得到的固定化漆酶最优,活力达到111.4 U·g-1;比较固定化漆酶和游离漆酶的性质发现,固定化漆酶p H稳定性和热稳定性均优于游离漆酶,固定化漆酶重复使用性好,与底物反复操作10次后,相对活性仍保持70.8%.     

漆树漆酶催化2,6-二甲基酚的氧化反应

在0.1mol/L磷酸盐缓冲液中,漆树漆酶催化2,6-二甲基酚(DMP)氧化生成3,3'5.5'-四甲基-4,4'-二苯醌(TMDQ).pH8.0,温度40～50℃时酶促氧化DMP较快.DMP浓度在1.66×10^-4～9.92×10^-4mol/L时为一级反应,速率常数为3.57×10^-5s^-1,共存的二茂铁(Fc-H)能大大促进酶促氧化DMP的速度.提出了该反应可能的反应机理。     

漆树漆酶的催化氧化作用(X)──叔丁基儿茶酚类底物的漆酶催化氧化和漆酶的微环境效应

对6个含叔丁基的儿茶酚类底物的离解和漆酶催化氧化反应进行了考察。结果表明:叔丁基的存在,有利于增进对底物的结合,并可以利用漆酶活性中心周围提供的pH环境和质子载体性质,促进底物的离解,使这类底物能在远低于其一级离解的弱酸性环境中进行反应。由于叔丁基的特性和漆酶的微环境效应的共同作用,这些底物在pH=6.0的水溶液环境中都能顺利地氧化成相应邻醌,并具有合成价值。ESR结果证明:这些底物在弱酸性条件下的漆酶催化反应经历了质子转移和单电子氧化步骤。     

漆酶/O2体系获取半醌自由基的研究

本文综述了在静态条件下用漆酶／O2体系获取高稳定浓度半醌自由基阴离子的有效办法。依据漆酶的单电子转移特性,分别采用强吸电子基团使未偶电子离域以稳定半醌自由基、适当的有机溶剂延缓半醌自由基衰减和ESR自旋稳定化技术三种途径。