Laccases are multi-copper oxidase enzymes that catalyze the oxidation of a broad range of chemical substrates while reducing O_2 directly to water. They are typically found in fungal organisms and plants, and are part of the ligninolitic consortium together with other oxidative enzymes that degrade lignin. This book provides new research on the applications, investigations and research insights on laccase.
Chapter 1 - The developments of laccase enzymes (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) in industrial applications and investigation are revised in this chapter. Laccases are considered as "green biocatalysts" and versatile enzymes for emerging biotechnological applications. Highlighting the key aspects and recent advances in biocatalysis, this book chapter also covers strategies for reaction mechanisms and for the enhancement of enzyme selectivity and efficiency, as well as technological advantages of laccase immobilization. Additionally, this book chapter reports suitable advanced technologies for laccase utilization on bioconversions in order to reduce costs and environmental impact. In fact, an increasing number of oxidative biocatalytic processes driven by laccases have been reported in the literature during the last two decades, emphasising the wide variability and potential of these enzymes. The chapter is divided into five main sections. A general introduction about laccases and their reaction mechanisms, structural properties, substrates, laccase mediator systems and their role in biocatalysis is presented. Laccase specific applications in pulp and paper, textile and food industries, organic synthesis, biorefineries, medical, biosensors and biofuel production as well as in bioremediation of phenolic compounds and wastewater treatment are described. The chapter finishes describing specific laccase immobilization on different types of supports, from natural fibres to nanoparticle materials and related applications. The authors also provide an important bibliographic revision related to the discussed topics. As a result, readers from academy, research institutes and industry may gain useful information and knowledge on the emerging laccase-based biocatalytic transformations.
Chapter 2 - Laccases are wide spread enzymes belonging to the family of the blue multi copper oxidases. Laccases posses the ability to oxidize a wide range of organic phenolic and non-phenolic compounds by a radical-catalyzed reaction mechanism, displaying both ligninolytic and polymerizing (cross-linking) abilities. This property allow these "old" biocatalysts to be applied in new biotechnological applications, making them as young as ever. As a fact, in the last decades, with the fast progress of enzyme technology, scientific and industrial world has been more and more interested in the exploitation of laccase in different application, as testified by the huge number of published research article and patents.
In this chapter, an overview of the most recent laccase applications is provided in order to underline the biotechnological importance of these enzyme. More in detail the role of laccase in surface grafting, in the construction of biosensing platforms, and in the degradation of different classes of synthetic pollutants such as dyes and EDCs is deeply investigated.
Chapter 3 - Biosensors are, by definition, sensing devices that include a biological component (enzyme, antibody, animal or plant cells, oligonucleotides, lipids, microorganisms, etc.) intimately connected to a physical transducer (electrode, optical fiber, vibrating quartz, etc.). This dual configuration permits the quantitative study of the interaction between target-molecules and these bioelements with high specificity and sensitivity. Typical recognition elements used in electrochemical biosensor platforms are enzymes; their selection and source have a major impact on the device performance. The interest in laccase-based electrochemical biosensors is due to laccase's biocatalyst ability and good stability. Thereby, laccase-based electrochemical biosensors have emerged as suitable tools for measuring the reduction current of quinones generated as products of the enzymatic reaction or based on the inhibition of the enzyme by the target analyte and other specific interactions in a simple, rapid, and economic way. They answer to the demands of food safety and quality, environment pollution control, pharmaceutical and clinical analysis, as well as to others fields. Moreover, laccase-based biosensors relying on electrochemical transduction are well suited because of their easy miniaturization and simple instrumentation. The proposed chapter presents a review of the available studies of the last five years on development and main areas of application of laccase-based electrochemical biosensors.
Chapter 4 - During the last years enzymatic fuel cells (EFCs) have attracted great interest due to their possible applications, especially as electrical power sources for implantable devices in living organisms. In EFCs enzymes are used as biocatalysts for fuel oxidation at the anode and oxidant reduction at the cathode. Depending on the fuel and oxidant available an appropriate choice of enzymes is needed for the biofuel application. The majority of EFCs use oxygen-reducing enzymes at the cathode, as it is a very common oxidant present in most human physiological fluids. Multi-copper oxidases, such as laccase, bilirubin oxidase, ascorbate oxidase, have been studied as possible biocatalysts for the oxygen reduction reaction; their main attractive comes in their skill to reduce oxygen directly to water avoiding H_2O_2 formation. In this review the authors focus on the use of laccase enzymes as biocatalyst for the fabrication of biocathodes, paying special attention to the different electrode materials and immobilization strategies used to manufacture biodevices based on direct electron transfer (DET) or on mediated electron transfer (MET). Indeed, a larger surface area of the support material allows higher enzyme loading, therefore increasing the current density developed. Nanoparticles, nanofibers, nanotubes, mesoporous and carbonaceous materials have been used for this purpose. A good immobilization strategy enhances the long-term stability of the biodevice while achieving efficient wiring of the enzyme.
Laccases usually exhibit higher activity at acid pH and they are strongly inhibited in the presence of chloride ions. As this could limit their use for possible implantable devices, a strategy has been made for overcoming this problem: native laccases have been engineered by directed evolution for obtaining mutants that show activity under physiological conditions. An alternative strategy followed has been altering the local pH of the biocathode.
Finally, future outlook of laccase-based biocathodes applications are highlighted.
Chapter 5 - Laccase has been regarded as one of the top bioelectrocatalysts for decades. Plenty of research has focused on the use of this enzyme together with electrodes to favour the oxygen reduction at high potential values. Biolectrocatalysis is conditioned to the enzyme structure: the catalytic site is usually buried inside a protein wrap specifically designed to optimize the enzyme's natural activity. This means that a typical biocatalyst such as laccase is anisotropic and its performance depends on its orientation after immobilization on the electrode. Therefore the chemical modification of the electrode surface with functional groups is crucial to achieve the best possible enzyme orientation. When preparing a laccase-modified electrode to work as O_2 biocathode two goals are sought: high current values and low overpotential. Low overpotential values can be obtained by orienting the laccase with its T1 site facing the electrode; enzymes non-properly oriented will not communicate with the electrode. Adding a mediator facilitates non-oriented laccases' connection with the electrode, increasing the current density provided by the biocathode at expenses of lowering the onset potential. In the present work the authors take a Coriolopsis gallica laccase and study its performance after two different covalent immobilization strategies for both direct electron transfer (DET) and ABTS-mediated electron transfer (MET). The first strategy comprises amine functionalization of the electrode and its coupling with the laccase acidic residues, the second strategy goes vice versa.
Chapter 6 - Laccase is commonly used enzyme in a cathode of biofuel cells to reduce molecular oxygen to water by providing four electrons. Since direct electron transfer between laccase and electrode due to spatial as well as electrochemical difficulty, appropriate mediators, typically metal complexes such as ferrocene, is used to enhance their functions. The authors searched for some types of metal complexes as mediators that have advantageous potential and sterical characters these candiates. This chapter describes syntheses of metal complexes, cyclic voltanmetry (CV) measurements in glassy carbon (GC) electrodes in acetate buffer, and compare their fuctions as mediator for laccase. The results of case studies are reported.
Chapter 7 - Laccases (benzenediol: oxygen oxidoreductase) are an interesting group of multi copper enzymes due to their ability to oxidize both phenolic and non-phenolic lignin related substrates as well as a wide spectrum of highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for application to several biotechnological processes including the bioremediation of contaminated soils and industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, polymer synthesis, wine and beverage stabilization; detoxification of lignocellulose hydrolysates for ethanol production; and construction of biosensors and biofuel cells. Industrial processes are often best employed at non-natural conditions that may include high temperature and/or pressure, high salt concentrations, acidic or alkaline pH, oxidative conditions, high shear forces or short delays. A major challenge of laccases technology is that the catalytic proficiency and stability of enzymes are dramatically dependent on environmental variables. Generally, fungal laccases are active at acidic pH and low ionic strengths, where the enzymes are much less stable. Thus, recent efforts have been directed both towards identifying novel extremophilic bacterial laccases with new catalytic properties for potential innovations and breakthroughs in industrial biotechnology. These extremozymes could be used in a number of future applications where high temperature or salinity, neutral or alkaline pH are desirable, including e.g., hair dying, organic synthesis, laundry detergence, some processes of bioconversion and bioremediation, etc. This chapter gives a current overview on the sources and characteristics of such extremophilic laccases, with particular emphasis on their strategy for function at harsh conditions and their potential industrial and biotechnological applications.
Chapter 8 - Laccase is one of the few enzymes studied from the end of the 19~th century, when it was found in the exudate from the Japanese lacquer tree (Rhus vernicifera). Later, laccases were also found in fungi, especially in those belonging to the ecological group of ligninolytic strains. Ligninolytic fungi possess unique physiological and biochemical properties, which permit them to transform and mineralize, in addition to their natural substrate lignin, a wide range of pollutants, including chlorinated compounds, synthetic dyes, and polycyclic aromatic hydrocarbons. The degradative activity of these fungi is due to a complex of extracellular oxidative enzymes, including laccase.
Laccases (p-diphenol oxidases, EC 1.10.3.2) belong to the family of multicopper oxidases that catalyze the oxidation of a range of aromatic substances with the concomitant reduction of O_2 to H_2O. They have overlapping substrate specificity, which can be extended to nonphenolic aromatic compounds, first by including redox mediators in the reaction mixture and second by modifying laccase's active center with lignin-degradation products. During solid-state fermentation of natural lignin-containing substrates, ligninolytic fungi produce a yellow form of laccase, in which the active center is modified by lignin-degradation products. As a result of this modification, laccase gains the ability to catalyze the oxidation of nonphenolic compounds without the addition of mediators. These unique catalytic properties of fungal laccases permit them to catalyze the key reactions in biodegradative processes such as the degradation of environmental xenobiotics.
This article reviews the most important molecular and catalytic properties of fungal laccases, including their molecular structure, substrate specificity, optimal pH and temperature values, need for effectors, and influence on laccase catalytic activity toward xenobiotics. The principal mechanisms of laccase-mediated degradation of lignin and xenobiotics, for example by coupling, are discussed. Finally, the potential of use of these enzymes, including their immobilized form, in biotechnology is considered.
Chapter 9 - In nature, laccases are involved in lignin synthesis but also in its degradation. This versatility, its relative unspecificity and potential to act on lignin, allow these oxidases to be applied in several industrial sectors. Among them, wood industry shows several scopes where laccases could be included in order to obtain new products, or conventional ones by greener manufacturing processes.
The manufacturing of wood composites is based in the use of different types of wood particles that are joined together by means of several adhesive formulations. Adhesives are mainly composed of chemicals from the petrochemical industry that are polymerized in contact with wood particles in order to form the composite. Laccases may help on this process by activating lignin and improving the adhesion process, resulting in the reduction of the conventional adhesive formulation, or even its absence.
Laccases are also able to mediate in the grafting of several monomers onto wood and, therefore, could be used as a sustainable tool to treat wood in order to provide different properties to this natural material. This enzymatic grafting is based on the initial formation of radical compounds from the laccase substrates, which are subsequently linked onto the wood by radical polymerization. By using this method stable bonds between enzymatic substrates and wood are formed and, consequently, leaching of the grafted compounds is avoided. The use of substrates with specific characteristics could be used to confer such features to the wood. Thus, some interesting properties such as hydrophobicity, durability, biofouling resistance, etc. could be stably provided by this kind of treatments.
Chapter 10 - Laccases are a group of multi copper oxidase enzymes of great interest due to their ability to degrade phenolic and non-phenolic compounds, while being biocompatible. Their capability of oxidation can be applied to detect and treat multiple chemical pollutants present in water resources and during food processing.
The presence of bioactive contaminants in drinking water have been considered a critical environmental issue due to its potential health and ecological effects. Within them, pollutants coming from pharmaceutical and personal care industries have increased the amount of endocrine disruptor compounds (EDCs) in water resources, leading to development problems in human beings and some other mammals. Lately, a laccase cocktail produced from Pycnoporus sanguineus CS43 fungi has shown to degrade different types of EDCs in groundwater samples.
In addition, different food processing methods often need to remove phenolic compounds from their products in order to achieve desired levels of quality. For instance, beer and wine stabilization use laccase cocktails as additives in their production process to remove polyphenol. Nevertheless, laccases are also of great interest for the detection of undesired compounds in food and beverages, regardless of their removal capability. Specifically, the development of enzyme-based biosensors is an interesting alternative for detection of pollutants. They provide selective quantitative or semi-quantitative analytical information through specific biochemical interactions.
The following chapter attempts to review the current state of art of the application of laccases for the monitoring and removal of water pollutants such as EDCs of anthropogenic origin, as well as the current and potential direct and indirect applications of laccase in the food industry.

Preface vii

Chapter 1 Laccase Properties, Reaction Mechanisms and Applications: An Overview 1

Chapter 2 New Trends in Laccase Applications 27

Chapter 3 Laccase-Based Biosensors for Electroanalysis: A Review 45

Chapter 4 Laccase-Based Biocathodes for Oxygen Electroreduction in Enzymatic Fuel Cells 75

Chapter 5 Immobilization and Electrochemical Characterization of Coriolopsis gallica High Redox Potential Laccase 101

Chapter 6 Enhancing Biofuel Cell Functions of Four Electron Oxygen-Reducing Laccase by Several Types of Metal Complexes as Madiators 111

Chapter 7 Biotchnological Potential of Extremophilic Laccases 125

Chapter 8 Unique Properties of Fungal Laccases for biodegradative processes 143

Chapter 9 Laccases for Wood Treatment: From Composites Manufacturing to Surface Modification 181

Chapter 10 Uses of Laccase in the Monitoring and Treatment of Water and Food 207

Index 231

中国医科院医学信息研究所