Metabisulfite interference in biosensing and Folin-Ciocalteu analysis of polyphenols

Sodium metabisulfite is one of the forms of sulphurous compounds which are added as preservative in food and beverage processing industries. Its interference with different methods of analysis of polyphenols was investigated in the present work. Studies involved the reaction of metabisulfite at platinum electrodes either at a constant applied potential of +100 mV vs. Ag/AgCl electrode with amperometric detection, or at linear potential sweep in cyclic voltammetry experiments. In a second stage, its effect on the analysis of polyphenols that are oxidised by enzymes was examined by the inhibition that metabisulfite caused on caffeic acid oxidation in the presence of free and immobilized laccase. It was found that immobilized laccase was less inhibited than free laccase. A confirmation of the influence of metabisulfite on the Folin-Ciocalteu analysis was also done.     

壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮-多壁碳纳米管复合物固定漆酶基化学传感器的性能

通过壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮(CTS-g-N-CSIDZ)非共价功能化多壁碳纳米管(MWCNTs)的方式制备固定漆酶载体,该复合物载体主要通过物理吸附和漆酶活性中心与载体上配体之间的配位作用来固定漆酶,较大程度地保持了游离漆酶活性位原始构象.将固定了漆酶的复合物附着在裸玻碳电极上便构筑了复合物固定漆酶修饰玻碳电极.在以分光光度法测定了这种复合物载体对漆酶的担载量、固定漆酶比活力、稳定性、重复使用性及其催化2,6-二甲氧基苯酚(DMP)氧化动力学参数的基础上,还对基于此种复合物固定漆酶修饰玻碳电极作为化学传感器(以DMP作为底物)的性能进行了研究.结果表明,该复合物具有较高的固酶担载量(81.7 mg/g)和固定漆酶比活力(1.33 U/mg);而作为电化学传感器的复合物固定漆酶修饰玻碳电极对底物DMP具有较高的亲和力(对DMP的米氏常数KM是0.0918 mmol/L),较高的灵敏度( 3680 mA· L/mol),较低的检测限(3.3×10-4 mmol/L),较高的响应选择性,良好的重现性、重复使用性和长期稳定性.这种漆酶基电极有望用作电流型特定结构的酚类传感器.     

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.     

Immobilization of laccase by Cu2+ chelate affinity interaction on surface modified magnetic silica particles and its use for the removal of pentachlorophenol

Magnetic Cu²⁺-chelated silica particles using polyacrylamide as a metal-chelating ligand was developed and used for the immobilization of laccase by coordination.The effect of pH and temperature on the enzymatic property of immobilized laccase and its catalytic capacity for pentachlorophenol（PCP） degradation were evaluated systemically.Compared with free laccase,the immobilized laccase showed improved acid adaptabihty and thermal stability.The immobilized laccase prepared in this work exhibited a good catalytic capacity for PCP removal from aqueous solutions.     

Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal

In this study, polyethyleneimine was combined with magnetic Fe₃O₄ nanoparticles through the bridging of carboxyl-functionalized ionic liquid, and laccase was loaded onto the carrier by Cu²⁺ chelation to achieve laccase immobilization (MCIL–PEI–Cu–lac). The carrier was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, thermogravimetric analysis, X-ray diffraction analysis, magnetic hysteresis loop and so on. MCIL–PEI–Cu–lac has good immobilization ability; its loading and activity retention could reach 52.19 mg/g and 91.65%, respectively. Compared with free laccase, its thermal stability and storage stability have been significantly improved, as well. After 6 h of storage at 60 °C, 51.45% of the laccase activity could still be retained, and 81.13% of the laccase activity remained after 1 month of storage at 3 °C. In the pollutants removal test, the removal rate of 2,4-dichlorophenol (10 mg/L) by MCIL–PEI–Cu–lac could reach 100% within 10 h, and the removal efficiency could still be maintained 60.21% after repeated use for 8 times. In addition, MCIL–PEI–Cu–lac also has a good removal effect on other phenolic pollutants (such as bisphenol A, phenol, 4-chlorophenol, etc.). Research results indicated that an efficient strategy for laccase immobilization to biodegrade phenolic pollutants was developed.     

Decolorization Potential of Some Reactive Dyes with Crude Laccase and Laccase-Mediated System

In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH 6) solution.     

Flavonoid-Rich Plants Used as Sole Substrate to Induce the Solid-State Fermentation of Laccase

High cost becomes the major obstacle for the industrial application of laccase. Many approaches have been applied to enhance the yield and decrease the cost of laccase. Since flavonoids are the natural inducers for laccase production, in this article, flavonoid-rich plants were taken as the sole substrate for the solid-state fermentation of Funalia trogii (Cui 3676). It indicated that flavonoid-rich plants can effectively promote the production of F. trogii laccase without the addition of inducers. The laccase activity was 42.5 IU g^?1 substrate when kudzu vine root was used as the substrate, which was enhanced by 4.46 times than that when bran was used as the substrate. Meanwhile, the solid-state fermentation of laccase could enrich flavonoids, benefiting their extraction. The content of flavonoids extracted from fermented kudzu vine root and Ginkgo biloba leaves was enhanced by 56.41 and 24.11 %, respectively, compared to the unfermented substrate, and the relative reductive ability and scavenging ability of hydroxyl radicals of flavonoids in the fermented residues were essentially unchanged. Thus, flavonoid-rich plants will become a kind of potential substrate for laccase fermentation which is beneficial in enhancing the yield and reducing the cost of laccase.     

Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

提高漆酶稳定性的化学修饰方法的研究

天然漆酶在其应用条件下容易失活,因此如何提高漆酶的稳定性显得尤为重要和紧迫.以目前广泛应用的商品化漆酶DeniLite ⅡS为研究对象,采用邻苯二甲酸酐(PA)、丁二酸酐(SA)及马来酸酐(MA)对漆酶进行化学修饰,根据修饰酶的酶活和热稳定性的变化确定SA为漆酶的最佳修饰剂.采用L9(34)正交设计表研究了磷酸盐缓冲液...     

Production and Characterization of a Novel Laccase with Cold Adaptation and High Thermal Stability from an Isolated Fungus

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml⁻¹, which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0°C but also a high thermostable enzyme. The half-lives at 60, 70 and 80°C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.     

Electrochemical Characteristics of Mediated Laccase‐Catalysis and Electrochemical Detection of Environmental Pollutants

Laccase has been immobilized on the carbon nanotubes modified glassy carbon electrode surface by adsorption. As‐prepared laccase retains good electrocatalytic activity to oxygen reduction by using 2,2′‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid) as the mediator. It can be used as a biosensor for the determination of catechol with broad linear range. Especially, azide, one of inhibitors of laccase, shows sensitive inhibition to catalytic activity of the laccase modified electrode. In addition, the inhibition by fluoride ions has also been studied. These demonstrate that the as‐prepared electrode can be used to detect halide and some the toxic pollutants, e.g., catechol and azide based on catalytic or inhibition reaction of laccase. The simple preparation procedure makes the system can be developed as non‐inhibition or inhibition biosensor.     

Determinants of the relative reduction potentials of type-1 copper sites in proteins

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.     

Electroenzymatic reactions with oxygen on laccase-modified electrodes in anhydrous (pure) organic solvent

The electroenzymatic reactions of Trametes hirsuta laccase in the pure organic solvent dimethyl sulfoxide (DMSO) have been investigated within the framework for potential use as a catalytic reaction scheme for oxygen reduction. The bioelectrochemical characteristics of laccase were investigated in two different ways: (i) by studying the electroreduction of oxygen in anhydrous DMSO via a direct electron transfer mechanism without proton donors and (ii) by doing the same experiments in the presence of laccase substrates, which display in pure organic solvents both the properties of electron donors as well as the properties of weak acids. The results obtained with laccase in anhydrous DMSO were compared with those obtained previously in aqueous buffer. It was shown that in the absence of proton donors under oxygenated conditions, formation of superoxide anion radicals is prevented at bare glassy carbon and graphite electrodes with adsorbed laccase. The influence of the time for drying the laccase solution at the electrode surface on the electroreduction of oxygen was studied. Investigating the electroenzymatic oxidation reaction of catechol and hydroquinone in DMSO reveals the formation of various intermediates of the substrates with different electrochemical activity under oxygenated conditions. The influence of the content of aqueous buffer in the organic solvent on the electrochemical behaviour of hydroquinone/1,4-benzoquinone couple was also studied.     

介孔SiO2/Fe3O4中空磁性微球的漆酶固定化

以介孔SiO2/Fe3O4磁性中空微球作为载体,采用物理吸附法对漆酶进行固定化,考察了时间、温度和pH值对漆酶固定化效果的影响,并对固定漆酶的活性及稳定性进行了研究.结果表明,介孔SiO2/Fe3O4磁性中空微球吸附漆酶分子后,介孔材料的比表面积与孔体积均减小.在3 h时复合微球对漆酶的吸附达到平衡,复合微球中介孔SiO2对漆酶的有效固定量为689 mg/g,大大高于纯介孔材料MCM-41的漆酶固定量(319 mg/g).在pH=3～6的条件下,复合微球中固定漆酶仍保持70%以上的相对酶活.当温度不高于60℃时,固定漆酶的相对酶活仍保持65%以上.固定漆酶的pH稳定性和热稳定性都明显优于游离漆酶,固定漆酶的米氏常数为1.05 mmol/L,与游离漆酶相比,固定漆酶与底物的亲和力有所降低.当2,4-二氯苯酚的浓度为10 mg/L时,固定漆酶对其去除率在6 h时达到81.6%,表现出很好的催化活性.     

漆酶在催化合成生物活性化合物中的研究进展

近年来市场对具有营养和药用价值的活性化合物的需求量逐年增加,传统的生产方法已无法满足该类化合物的大规模应用。漆酶是近些年广受欢迎的生物催化剂之一,它可以在温和的条件下催化活性化合物的高效合成,并且有极大潜力取代传统的工业生产方法。本文着重回顾了近十年来漆酶在催化合成活性化合物中的应用,并对漆酶的结构及作用机制进行了介绍;同时指出了漆酶工业化应用中存在的一些问题,比如漆酶产量不足、部分酶促反应介质不适于工业化应用等。通过异源表达、筛选高产菌株提高漆酶产量、使用固定化技术和蛋白质工程提高漆酶的使用寿命、开发更加高效低廉的反应介质系统与寻找新的漆酶底物相结合来降低漆酶的应用成本是今后主要的发展趋势。     

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.     

Utilization of Horticultural Waste for Laccase Production by <Emphasis Type="Italic">Trametes versicolor</Emphasis> Under Solid-State Fermentation

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.     

漆树酶固定化及其性质研究

本文介绍了用过渡金属水合氧化物固定化漆树漆酶的方法。考察了整合法固定漆树酶的最适条件。对固定化前后漆酶的性质进行了比较,并分析了造成固定化酶与游离酶性质差异的原因。     

大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。