Laccase: a multi-purpose biocatalyst at the forefront of biotechnology.

Our current global environmental challenges include the reduction of harmful chemicals and their derivatives. Bioremediation has been a key strategy to control the massive presence of chemicals in the environment. Enzymes including the phenoloxidases, laccases and tyrosinases, are increasingly being investigated as “green products” in the removal of many chemical contaminants in waters and soils. Both phenoloxidases are widespread in nature and attractive biocatalysts due to their ability to use readily available molecular oxygen as sole cofactor for their catalytic elimination of a large number of chemicals. Taking advantage of their catalytic potentials, remarkable advances have been made in the engineering of laccases to produce suitable biocatalysts in environmental applications. Studies about novel strategies of laccase immobilization and insolubilization for the treatment of chemical contaminants were provided. Likewise, tyrosinases are gaining increasing interest in environmental applications due to their catalytic similarities with laccases although they remain far less investigated to date. This disparity was addressed in this review along with the molecular features and catalytic mechanism of tyrosinases relevant in environmental applications. A perspective on the future use of laccases and tyrosinases in bioremediation was discussed.

The ever-increasing depletion of fossil fuels and the need for clean methods of producing electricity have stimulated the emergence of abiotic and enzyme-based fuel cells that convert chemical energy into electrical energy at room temperature [1]. Taking into account that the interface activity was related to the immobilized amount of catalysts and the specific surface of the conductive substrate, 3D structures were designed via the use of carbon nanotubes as building block [2]. Carbon nanotubes exhibit nanowire morphology, biocompatibility and excellent conductivity. Furthermore, nanotube modified electrodes offer a large electroactive surface together with a highly porous three-dimensional structure. The recent advances in term of enzyme immobilization via functionalized films electropolymerized onto nanotube coatings will be described. For instance, the combination of carbon nanotube coating and electropolymerized photoreactive films constitutes an original strategy for the covalent immobilization of proteins by irradiation. We describe, here, the successful electrogeneration of a photoactivatable polypyrrole-diazirine film onto multi-walled carbon nanotube coatings and its use for the covalent binding of proteins [3].Moreover, the oriented immobilization of laccase on carbon nanotube was carried out to enhance the direct electron transfer. The possibility to functionalize carbon nanotubes by attaching specific docking sites for enzymes via electrogenerated polymer or pyrene derivatives exhibiting p-stacking interactions will be reported [4,5].We report also the first example of a glucose/H2O2 biofuel cell operating at 5 mM glucose under air (Figure 1). At a bienzymatic cathode, the direct wiring of horseradish peroxidase achieves the electrocatalytic reduction of H2O2 produced during the oxidation of glucose [6]. This represents a novel alternative to the use of laccases or bilirubin oxidases in conventional glucose/O2biofuel cells for implantable applications.Taking into account that conductive nanostructured materials such as carbon nanotubes became highly appropriate candidates for the elaboration of biofuel cells and electrochemical supercapacitors, we report here for the first time, the original combination of these two devices. The possibility to recharge supercapacitors with an internal energy source could thus represent a significant improvement for the performance of biofuel cells. In this context, we propose an original hybrid battery-capacitor system using a compression of enzymes-carbon nanotubes as supercapacitors and electrodes for a biofuel cell setup (Figure 1). This hybrid supercapacitor/biofuel cell enables high power discharge cycles, the carbon nanotube disks being continuously recharged through the biocatalytic energy conversion under physiological conditions [7].Figure 1: Schematic presentation of the functioning principle of a glucose-H2O2biofuel cell and scheme of the electrochemical double layer supercapacitor – biofuel cell hybrid system.

Hazardous contaminants in the environment and their laccase-assisted degradation – A review

1,10-Phenanthroline derivatives as mediators for glucose oxidase

Improving the performance of lactate/oxygen biofuel cells using a microfluidic design

Laccases [EC 1.10.3.2, para‐bezenediol:dioxygen oxidoreductases] are multi‐copper proteins that oxidize various aromatic and non‐aromatic compounds by a radical‐catalyzed reaction mechanism using molecular oxygen as the secondary substrate. Laccase has a number of biological roles that include lignification, delignification, turnover of humic substances in soil and aquatic environments, pathogenicity, detoxification, morphogenesis, sporulation, growth and development of rhizomorphs, polymerization of melanin precursors and spore coat resistance. The ability of laccase to catalyze oxidation with only oxygen as a secondary substrate, its broad substrate range, and high catalytic constants have made it an appealing enzyme for a variety of remediative applications. These include bioremediation of phenolic‐rich wastewaters generated by paper and pulp, textile and dye, food and distillery industries as well as bioremediation of soils contaminated with recalcitrant xenobiotics such as chlorinated phenolic compounds, polycyclic aromatic hydrocarbons, explosives, endocrine‐disrupting chemicals, insecticides, fungicides, and herbicides. The enzyme's broad substrate spectrum, the use of readily available oxygen as the final electron acceptor, and no requirement for cofactors or peroxide have made it appealing for a variety of industrial applications in fields ranging from textile dyes, pulp and paper, biosensors, biofuel cells, chemical synthesis, beverage stabilisation, food products, and ethanol production. The advantages of laccase compared to other enzymes include relatively high yields, uncomplicated isolation from bulk fungal cultures, easy screening for specific producers with a broad substrate spectrum, a high degree of stability and activity especially after immobilization, and heterologous expression systems that have already been established. The characteristics of laccase allow for its potential utilization across a number of research areas, which will result in continued intensive investigations to better understand the enzyme and apply it forthe benefit of both humans and the environment.

Laccase, an incredible enzyme, has a wide prospective in bioremediation processes, mainly due to its relative broad oxidation capacity, the lack of requirement of cofactors, and the use of readily available oxygen as the final electron acceptor. However, the large-scale application of laccases in bioremediation necessitates immobilization/insolubilization of the biocatalysts to enhance their operational stability. With the burgeoning use of laccases in wastewater treatment, several state-of-the-art methods have been developed over the past few years to immobilize laccase, derived from various microbial sources, in order to enhance the selectivity, activity, stability, and reusability. Recent advances in these immobilization methods offer promising solutions to the limitations of soluble enzymes, such as poor reusability due to poor recoverability, low stability, and high costs, to name a few. This article is intended to review the various recent methods employed for immobilization or insolubilization of laccase and its use in treating various types of organic contaminants in wastewaters including those from olive mill, pulp and paper, biorefinery, municipal, hospital, and textile industries. Furthermore, to improve the potential of the laccase-based biocatalytic system against wastewater/pollution treatment, co-immobilization of enzymes such as tyrosinase, peroxidase, and glucose oxidase, with laccase, would serve as a promising bioremediation tool for treating the organic contaminants in industrial and municipal wastewater. The concept and approach of this review also renders knowledge on a yet unexplored focus on the pioneering advances on the development of immobilized laccase-based reusable biocatalysts, which could be employed for treatment of industrial and hospital wastewater.

This work presents the development and characterization of a self-powered electrochemical lactate biosensor for real-time monitoring of lactic acid. The bioanode and biocathode were modified with D-lactate dehydrogenase (D-LDH) and bilirubin oxidase (BOD), respectively, to facilitate the oxidation and reduction of lactic acid and molecular oxygen. The bioelectrodes were arranged in a parallel configuration to construct the biofuel cell. This biofuel cell’s current–voltage characteristic was analyzed in the presence of various lactic acid concentrations over a range of 1–25 mM. An open circuit voltage of 395.3 mV and a short circuit current density of 418.8 µA/cm² were obtained when operating in 25 mM lactic acid. Additionally, a 10 pF capacitor was integrated via a charge pump circuit to the biofuel cell to realize the self-powered lactate biosensor with a footprint of 1.4 cm × 2 cm. The charge pump enabled the boosting of the biofuel cell voltage in bursts of 1.2–1.8 V via the capacitor. By observing the burst frequency of a 10 pF capacitor, the exact concentration of lactic acid was deduced. As a self-powered lactate sensor, a linear dynamic range of 1–100 mM lactic acid was observed under physiologic conditions (37 °C, pH 7.4) and the sensor exhibited an excellent sensitivity of 125.88 Hz/mM-cm2. This electrochemical lactate biosensor has the potential to be used for the real-time monitoring of lactic acid level in biological fluids.

Enzymatic electrodes for glucose biosensors and glucose/oxygen biofuel cells were prepared by the sequential coating of carbon nanotube (CNT), ZnO nano rods, charge transfer complex based on tetracyanoquinodimethane and tetrathiafulvalene (TCNQ-TTF), and glucose oxidase. Among the prepared electrodes (TCNQ-TTF/GOx/Nafion, ZnO/GOx/Nafion, CNT/GOx/Nafion, ZnO/TCNQ-TTF/GOx/Nafion, and CNT/TCNQ-TTF/GOx/Nafion), the CNT/TCNQ-TTF/GOx/Nafion electrode exhibited the best electrochemical performance. It was found that the TCNQ-TTF electron mediator played a critical role in the electron transfer for the active sites of GOx to the electrode and the peak current increased by 150% due to the presence of CNTs. The peak current of the CNT/TCNQ-TTF/GOx/Nafion electrode increased linearly with the increase of glucose concentration in the range of 0-15 mM. The CNT/TCNQ-TTF/GOx/Nafion electrode was integrated with a bilirubin oxidase-immobilized cathode for biofuel cell applications. The maximum power density at glucose concentrations of 20 and 200 mM were 8.1 and 17.8 microW/cm2, respectively. The result of this study indicates that the CNT/Fc/GOx/CHI electrode can be applied in the development of biofuel cells and biosensors.

Laccase engineering: From rational design to directed evolution

Laccases are multicopper-containing enzymes that have the ability to oxidize a wide variety of substrates with a single electron transfer reaction. These are environmentally benign versatile biocatalysts that have gained great interest in the biotechnological community since they utilize molecular oxygen as the last electron acceptor and only produce water as a byproduct. This family of enzymes has been widely used in a broad variety of applications, ranging from food additives and beverage processing to biological diagnostics and even as crosslinking agents in the furniture construction and manufacture of biofuels. Considering the benefits of enzyme immobilization, there has been a dramatic increase in applying immobilized laccases in recent years. Despite the impressive biotechnological promise, the use of laccases in the real world is still constrained by cost–benefit analysis, particularly in terms of practically large-scale production. The enzyme industry is booming research on laccase production, and use neglects to include the economic impact of the operations. Because of their ability to metabolize complex xenobiotics, they are also useful biocatalysts in enzymatic bioremediation processes, such as wastewater treatment. This study discusses the most important and recent breakthroughs in the biocatalytic attributes, sources, and exploitation of laccases in biotechnology for a sustainable industry.

Electrochemical methods based on enzyme-electrochemical reactions have been developed for studying oxidoreductase reactions. The methods measure a current resulting from an oxidoreductase reaction with an electrode serving as a final electron acceptor (or donor) in the reaction. A theoretical equation for the enzyme-electrochemical reaction, called bioelectrocatalysis, is derived, which enables kinetic analysis of the reaction. In combination with spectrophotometry, the electrochemical method provides a method for determining the redox potentials of proteins and enzymes. An alternative method based on bulk electrolysis in a quartz cell for UV-vis spectroscopy has been developed for the measurements of protein redox potentials on a conventional spectrophotometer. The electrochemical methods are applied to kinetic and thermodynamic analyses for the reactions of a variety of enzymes including a newly discovered enzyme, quinohemoprotein amine dehydrogenase (QH-AmDH), and bilirubin oxidase (BOD) [EC 1.3.3.5, from Myrothecium verrucaria], a copper-containing enzyme useful for bioelectrocatalytic O(2) reduction in biofuel cells. The electrochemical method for kinetic analysis has been successfully applied to the analysis of oxidoreductase reactions in vivo, as demonstrated by the reaction of glucose dehydrogenase in Escherichia coli. The advantages of the electrochemical methods are discussed.

Bioenergy and biofuels are promising candidates as alternative fossil fuel. Nanotechnology has been accepted as important tool for the synthesis and modification of bioenergy and biofuel production. In this chapter, we have focused on the recent development of carbon-based nanomaterials in the applications of biofuel cell. Obviously, carbon-based nanomaterials could be obtained from biomass, which possess the properties of nanomaterials, implying the complete combination of biofuel and nanotechnology. We have described the preparation of carbon-based nanomaterials with various structures and shapes by all kinds of synthesis methods. It is well known that carbon-based nanomaterials have wide applications in photocatalysis, biomedical, sensor, etc. Special attention has been paid on the potential applications of carbon-based nanomaterials in the biofuel cell. We also suggested the problems and future developments of carbon-based nanomaterials in these special fields. In this chapter, our purpose was to review the development and problems on the carbon-based nanomaterials in biofuel cell application.

Direct electron transfer (DET) is a unique feature of some enzymes. The possibility of DET between enzymes and the electrode surface could pave the way for superior reagentless, noncompartmentised, mediator‐free biofuel cells, as it obviates the need for mediators and allows an efficient transduction of the electrical current. DET is highly beneficial in the development of enzymatic and microbial biofuel cells. In this review article, hemoproteins, which are able to directly transfer electrons to the surfaces of conducting supports, are briefly overviewed and characterised. The main focus is laid on the application of heme‐c containing enzymes in biofuel cell design. Some historical steps and recent developments in biofuel cell design are presented in this article. Various designs of biofuel cells are overviewed. Possible applications of biofuel cells are presented and/or predicted and discussed. Problems and challenges in biofuel cell design and application are identified while possible directions to solve recent problems in biofuel cell development are discussed. The application of enzymatic biofuel cells as model systems and tools for advanced study of bioelectronics' properties of enzymes is predicted.

This review focuses on the development of microbial biofuel cells to demonstrate how similar principles apply to the development of bioelectronic devices. The low specificity of microorganism-based amperometric biosensors can be exploited in designing microbial biofuel cells, enabling them to consume a broader range of chemical fuels. Charge transfer efficiency is among the most challenging and critical issues while developing biofuel cells. Nanomaterials and particular redox mediators are exploited to facilitate charge transfer between biomaterials and biofuel cell electrodes. The application of conductive polymers (CPs) can improve the efficiency of biofuel cells while CPs are well-suitable for the immobilization of enzymes, and in some specific circumstances, CPs can facilitate charge transfer. Moreover, biocompatibility is an important issue during the development of implantable biofuel cells. Therefore, biocompatibility-related aspects of conducting polymers with microorganisms are discussed in this review. Ways to modify cell-wall/membrane and to improve charge transfer efficiency and suitability for biofuel cell design are outlined.