Bioelectrocatalytic properties of lignin peroxidase from Phanerochaete chrysosporium in reactions with phenols, catechols and lignin-model compounds

Bioelectrocatalytic reduction of H(2)O(2) catalysed by lignin peroxidase from Phanerochaete chrysosporium (LiP) was studied with LiP-modified graphite electrodes to elucidate the ability of LiP to electro-enzymatically oxidise phenols, catechols, as well as veratryl alcohol (VA) and some other high-redox-potential lignin model compounds (LMC). Flow-through amperometric experiments performed at +0.1 V vs. Ag|AgCl demonstrated that LiP displayed significant bioelectrocatalytic activity for the reduction of H(2)O(2) both directly (i.e., in direct electron transfer (ET) reaction between LiP and the electrode) and using most of studied compounds acting as redox mediators in the LiP bioelectrocatalytic cycle, with a pH optimum of 3.0. The bioelectrocatalytic reduction of H(2)O(2) mediated by VA and effects of VA on the efficiency of bioelectrocatalytic oxidation of other co-substrates acting as mediators were investigated. The bioelectrocatalytic oxidation of phenol- and catechol derivatives and 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulphonate) by LiP was independent of the presence of VA, whereas the efficiency of the LiP bioelectrocatalysis with the majority of other LMC acting as mediators increased upon addition of VA. Special cases were phenol and 4-methoxymandelic acid (4-MMA). Both phenol and 4-MMA suppressed the bioelectrocatalytic activity of LiP below the direct ET level, which was, however, restored and increased in the presence of VA mediating the ET between LiP and these two compounds. The obtained results suggest different mechanisms for the bioelectrocatalysis of LiP depending on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of LiP in biotechnological processes as solid-phase bio(electro)catalyst for decomposition/detection of recalcitrant aromatic compounds.     

Bioelectrocatalytic properties of lignin peroxidase from Phanerochaete chrysosporium in reactions with phenols,catechols and lignin-model compounds

Bioelectrocatalytic reduction of H₂O₂ catalysed by lignin peroxidase from Phanerochaete chrysosporium (LiP) was studied with LiP-modified graphite electrodes to elucidate the ability of LiP to electro-enzymatically oxidise phenols, catechols, as well as veratryl alcohol (VA) and some other high-redox-potential lignin model compounds (LMC). Flow-through amperometric experiments performed at +0.1 V vs. Ag|AgCl demonstrated that LiP displayed significant bioelectrocatalytic activity for the reduction of H₂O₂ both directly (i.e., in direct electron transfer (ET) reaction between LiP and the electrode) and using most of studied compounds acting as redox mediators in the LiP bioelectrocatalytic cycle, with a pH optimum of 3.0. The bioelectrocatalytic reduction of H₂O₂ mediated by VA and effects of VA on the efficiency of bioelectrocatalytic oxidation of other co-substrates acting as mediators were investigated. The bioelectrocatalytic oxidation of phenol- and catechol derivatives and 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulphonate) by LiP was independent of the presence of VA, whereas the efficiency of the LiP bioelectrocatalysis with the majority of other LMC acting as mediators increased upon addition of VA. Special cases were phenol and 4-methoxymandelic acid (4-MMA). Both phenol and 4-MMA suppressed the bioelectrocatalytic activity of LiP below the direct ET level, which was, however, restored and increased in the presence of VA mediating the ET between LiP and these two compounds. The obtained results suggest different mechanisms for the bioelectrocatalysis of LiP depending on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of LiP in biotechnological processes as solid-phase bio(electro)catalyst for decomposition/detection of recalcitrant aromatic compounds.     

Bioelectrocatalytic application of titania nanotube array for molecule detection

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60 nm and total length of 540 nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0 × 10⁻⁴ mM. A biosensor based on the glucose oxidase–titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (−0.4 V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6 s and a detection limit of 2.0 × 10⁻³ mM. The corresponding detection sensitivity was 45.5 μA mM⁻¹ cm⁻². A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.     

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.     

Bioelectrocatalysis-based application of quinoproteins and quinoprotein-containing bacterial cells in biosensors and biofuel cells

Electrochemical studies on the applied aspects of quinoproteins are briefly reviewed. Catalytic reactions of quinoprotein enzymes can be connected to electrochemical reactions directly or by the mediation of molecules functioning as electron acceptors of the enzymes. Such an enzyme-electrochemical reaction is called bioelectrocatalysis. It provides a novel method of kinetic analysis of enzyme catalysis and even whole bacterial cell catalysis. The principle of bioelectrocatalysis is first described, then, the bioelectrocatalysis-based application of quinoproteins in biosensors is mentioned. Characteristics and performance of this type of biosensor is explained by citing our own work. Possible application in bioreactors and biofuel cells is also mentioned.     

Cerbinal,a Pseudoazulene Iridoid,as a Potent Antifungal Compound Isolated from Gardenia jasminoides Ellis

d-Gluconate dehydrogenase isolated from Pseudomonas fluorescens was immobilized on the surfaces of carbon and gold electrodes by irreversible adsorption. The electrodes with the adsorbed enzyme produced anodic currents in solutions containing d-gluconate. The currents were attributable to the electro-enzymic oxidation (direct bioelectrocatalytic oxidation) of d-gluconate; the electrochemical system required no external redox molecules serving as mediators of electron transfer between the electrode and the adsorbed enzyme. A model of the direct bioelectrocatalysis at the enzyme-modified electrodes is presented.     

A computational protocol to predict suitable redox mediators for substitution of NAD(P)H in P450 monooxygenases

P450s catalyze a wide spectrum of stereo- and regioselective reactions like hydroxylations, epoxidations and dehydrogenations. Therefore biotransformations with P450s are of great relevance to organic synthesis. The use of isolated enzymes offers advantages over the use of whole cells. A key issue for catalytic applications of isolated P450s is the demand for a continuous electron supply to the heme-group. Mediator driven bioelectrocatalysis can help to overcome this problem.For mediator driven bioelectrocatalysis the identification of a suitable mediator is crucial for the fast development of an efficient electro enzymatic process. To this end we have developed a computational screening method based on using freely available software. Calculated electron transfer rates were compared with measured product formation rates. The novel in silico procedure allows a faster identification of suitable mediators for electrochemically driven P450 catalyzed reactions and can be used as screening tool. It may also lead to a massive reduction of experimental effort for the development of bioelectrochemical reaction systems in the future.     

Bioelectric power generation

In the recent article by Chaudhuri and Lovley, a fuel cell is described in which a microorganism, Rhodoferax ferrireducens, is used to oxidize glucose to carbon dioxide at neutral pH. This reaction occurs via direct bioelectrocatalysis: the microorganism uses the anode itself as the terminal oxidant to which liberated electrons are transferred, and does so with 83% efficiency. These findings are significant because they demonstrate a new approach for harvesting energy from the environment using microorganisms.     

Catecholamine detection using enzymatic amplification

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0·5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.     

Studies on the electrochemical performance of glucose biosensor based on ferrocene encapsulated ORMOSIL and glucose oxidase modified graphite paste electrode

The electrochemical performance of a new glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) and ferrocene encapsulated palladium (Pd)-linked organically modified sol-gel glass (ORMOSIL) material incorporated within graphite paste electrode. The ORMOSIL material incorporated within graphite paste electrode behaves as an excellent electrocatalyst for the oxidation of enzymatically reduced GOD. The electrochemical behavior of new glucose biosensor has been examined by cyclic volammetry and amperometric measurements. The bioelectrocatalysis of ORMOSIL embedded within graphite paste as a function of storage time and varying concentration of ORMOSIL is reported. The initial amperometric response on glucose sensing is recorded to be 145 microA at 15% (w/w) concentration of the ORMOSIL which is decreased to 20 microA at 5% of the same keeping GOD concentration constant. The variation of electrochemical behavior of the ORMOSIL embedded within graphite paste as a function of time has also been studied based on cyclic voltammetry. The voltammograms showing the reversible electrochemistry of ORMOSIL encapsulated ferrocene is changed into a plateau shape as a function of time, however, the electrocatalytic behavior is still retained. The practical usability of new glucose sensor has been compared with earlier developed glucose sensor. The sensitivity, response time and linearity of the new glucose biosensor are found to be excellent over earlier reported glucose biosensor. The amperometric response, calibration curve and practical applications of new glucose sensor are reported.     

Molecularly Ordered Bioelectrocatalytic Composite Inside a Film of Aligned Carbon Nanotubes

Molecularly ordered composites of polyvinylimidazole‐[Os(bipyridine)₂Cl] (PVI‐[Os(bpy)₂Cl]) and glucose oxidase (GOD) are assembled inside a film of aligned carbon nanotubes. The structure of the prepared GOD/PVI‐[Os(bpy)₂Cl]/CNT composite film is entirely uniform and stable; more than 90% bioelectrocatalytic activity could be maintained even after storage for 6 d. Owing to the ideal positional relationship achieved between enzyme, mediator, and electrode, the prepared film shows a high bioelectrocatalytic activity for glucose oxidation (ca. 15 mA cm^?2 at 25 °C) with an extremely high electron‐transfer turnover rate (ca. 650 s^?1) comparable to the value for GOD solutions, indicating almost every enzyme molecule entrapped within the ensemble (ca. 3 × 10¹² enzymes in a 1 mm × 1 mm film) can work to the fullest extent. This free‐standing, flexible composite film can be used by winding on a needle device; as an example, a self‐powered sugar monitor is demonstrated.     

Energizing cell-free protein synthesis with glucose metabolism

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.     

Electrochemical properties of polyaniline/carboxydextran (PANI/carDEX) composite films for biofuel cells in neutral aqueous solutions

Electrochemical properties of composite films consisting of polyaniline/carboxydextran (PANI/carDEX) as a biofuel cell electrode platform were investigated. These composite films were formed on a planar gold surface through electropolymerization after a simple chemical modification of dextran with carboxyl groups. Cyclic voltammetry indicated that the composite films retained a redox activity in neutral pH environment. The PANI/carDEX composite films showed an electrocatalytic activity for the oxidation of ascorbic acid. The PANI/carDEX composite films also demonstrated an excellent electron-transfer mediating capability for the bioelectrocatalytic activation of glucose oxidase (GOx) toward the oxidation of glucose.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

Improvement of the anodic bioelectrocatalytic activity of mixed culture biofilms by a simple consecutive electrochemical selection procedure

In this paper we demonstrate that the anodic, bioelectrocatalytic performance of wastewater inoculum based, mixed culture microbial biofilms can be considerably improved by using a consecutive, purely electrochemical selection and biofilm acclimatization procedure. The procedure may represent an alternative to a repetitive mechanical biofilm removal, re-suspension and electrochemically facilitated biofilm formation. By using the proposed technique, the bioelectrocatalytic current density was increased from the primary to the secondary biofilm from 250 microAcm(-2) to about 500 microAcm(-2); and the power density of respective microbial fuel cells could be increased from 686 mWm(-2) to 1487 mWm(-2). The electrochemical characterization of the biofilms reveals a strong similarity to Geobacter sulfurreducens biofilms, which may indicate a dominating role of this bacterium in the biofilms.     

生物电化学及其在环境监测中的应用

简要概述了生物电化学的研究领域,包括生物体系和生物界面模拟、生物分子的电化学、生物电催化、光合作用模拟和活组织电化学;总结了生物电化学传感器、生物芯片和生物电化学反应器在环境监测中的应用现状,并提出了其发展趋势,即不断向商品化方向发展,实现环境污染物的在线检测;利用基因技术,创造出检测能力更强的生物传感器和生物芯片;与其他精密分析仪器相结合,向多功能、集成化、智能化、微型化方向发展。     

微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Carbon nanofiber-based composites for the construction of mediator-free biosensors

Carbon nanofibers (CNFs), with typical diameters of approximately 80 nm and lengths of the order of micrometers, are extremely attractive in bioanalytical area as they can combine properties of high surface area, non-toxicity, acceptable biocompatibility, ease of fabrication, chemical and electrochemical stability, good electrical conductivity. In this work, CNF-based composites were successfully used as an immobilization matrix for the construction of a reagentless mediator-free hemoglobin-based H2O2 biosensor. The results revealed that hemoglobin retained its essential secondary structure in the CNF-based composite film. With the advantages of organic-inorganic hybrid materials, dramatically facilitated direct electron transfer of hemoglobin and good bioelectrocatalytic activity towards H2O2 were demonstrated. The biosensor displayed good performance along with good long-term stability. The CNF-based composites were proved to be a promising biosensing platform for the construction of mediator-free biosensors, and may find wide potential applications in biosensors, biocatalysis, bioelectronics and biofuel cell.     

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35°C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification products and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35°C. There was no significant accumulation (<250 μg) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35°C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.