Bacterial Laccases: Engineering, Immobilization, Heterologous Production, and Industrial Applications

Bacterial Laccases: Engineering, Immobilization, Heterologous Production, and Industrial Applications provides a list of approaches that upgrades bacterial laccases to industrially relevant enzymes. Providing protocols for enzyme production and downstream processing, and including up-to-date information on bacterial laccases, as well as techniques that can be explored to enhance properties and production levels, this book clarifies research gaps and explains how they can be dealt with. Written for research scholars working on enzymes, this is a valuable tool for industries and policymakers.

• Leads to a better understanding of traditional and novel methodologies for enhancing production and properties of bacterial laccases
• Serves as a useful guide for researchers, industrialists and students
• Includes chapters written by experts known for their contributions in respective areas
• Describes the latest advances made in the field of bacterial laccases, including strategies that could be undertaken to improve their utility at an industrial level
• Provides a comprehensive, up-to-date review of bacterial laccases and their important applications

• Language: English
• Publisher: Academic Press
• Release date: Nov 14, 2023
• ISBN: 9780323914574

Book preview

Front Cover for Bacterial Laccases - Progress in Biochemistry and Biotechnology - 1st edition - by Deepti Yadav, Tukayi Kudanga

Bacterial Laccases

Progress in Biochemistry and Biotechnology

Edited by

Deepti Yadav

Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Tukayi Kudanga

Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Table of Contents

Cover image

Title page

Copyright

Dedication

List of contributors

Preface

Chapter 1. Bacterial laccases: a general introduction

Abstract

1.1 Introduction

1.2 Laccase active site and reaction mechanism

1.3 Laccase sources

1.4 Brief review of applications of laccases

1.5 Prospects for bacterial laccases

1.6 Conclusion

References

Chapter 2. Agro-industrial wastes in bacterial laccase production by submerged and solid-state fermentation

Abstract

2.1 Introduction

2.2 Mode of action of laccases

2.3 Industrial importance of bacterial laccases

2.4 Bioprocesses for laccase production using agro-industrial wastes

2.5 Laccase production by solid-state fermentation

2.6 Laccase production by submerged fermentation

2.7 Economics of enzyme production

2.8 Conclusion

References

Chapter 3. Recombinant laccase production: Escherichia coli, Pichia pastoris, and filamentous fungi as microbial factories

Abstract

3.1 Introduction

3.2 Strategies for efficient recombinant protein production

3.3 Different strategies for recombinant laccase production

3.4 Conclusion

References

Chapter 4. Challenges in recovery and purification of laccases

Abstract

4.1 Introduction

4.2 Recovery and purification of extracellular and intracellular laccases

4.3 Challenges in the recovery and purification of laccases

4.4 Preferred solutions to the challenges of laccase recovery and purification

4.5 Conclusion

References

Chapter 5. Laccase engineering: tailoring laccases for effective and efficient catalysis

Abstract

5.1 Introduction

5.2 Mechanism of action

5.3 Structure and catalysis

5.4 Structure-guided rational design of the substrate specificity and catalytic activity of an enzyme

5.5 Approaches to alter laccase properties

5.6 Protein engineering: the search for a robust biocatalyst

5.7 Elucidation of the structure-function relationship of modified enzymes by in-silico tools

5.8 OB-1 Laccases

5.9 Consensus design of OB-1 laccases

5.10 Advantages of engineered laccases over natural laccases

5.11 Improvement in substrate specificity and catalytic activity

5.12 Enhancement of heterologous expression levels

5.13 Enhancement of stability of laccases

5.14 Conclusion

References

Further reading

Chapter 6. Immobilization for enhancement of laccase reusability

Abstract

6.1 Immobilization: trapping enzymes

6.2 Immobilization of bacterial laccases

6.3 Matrices for immobilization

6.4 Arsenal of bacterial laccases: future perspectives and novel applications

6.5 Conclusion

References

Chapter 7. Emerging contaminants and their possible bioremediation through bacterial laccases

Abstract

7.1 Introduction

7.2 Emerging contaminants and their risks

7.3 Treatment methodologies for emerging contaminants

7.4 Enzymes applied in bioremediation of emerging contaminants

7.5 Bacterial laccases applied for the bioremediation of emerging contaminants

7.6 Concluding remarks

Statements and declarations

References

Chapter 8. Bacterial laccases as versatile catalysts in material surface functionalization

Abstract

8.1 Introduction

8.2 Laccase synthesis of surface coatings

8.3 Laccase synthesis of dyes

8.4 Laccase in hair dyeing: synthesis and formulations

8.5 Laccases as bioactive oxygen scavenging coating barriers

8.6 Laccase application in increasing biobased material surface bonding

8.7 Laccases in pulp and paper processing

8.8 Laccase-mediated surface modification of synthetic polymers

8.9 Conclusions and future perspectives

References

Chapter 9. Bacterial laccase-like multicopper oxidases in delignification and detoxification processes

Abstract

9.1 Introduction

9.2 Lignocellulose as a renewable resource

9.3 Lignocellulosic biorefineries

9.4 Pulp and paper industry

9.5 Bacterial multicopper oxidases and their biochemical properties

9.6 The laccase mediator system

9.7 Application of bacterial multicopper oxidases in delignification and detoxification processes

9.8 Taking the next step: finding the best-fit bacterial multicopper oxidase for lignocellulosic biorefineries

9.9 Conclusion

Acknowledgements

References

Chapter 10. Laccases in organic synthesis

Abstract

10.1 Introduction

10.2 Laccase historical timeline

10.3 Synthesis of bioactive compounds

10.4 The future of laccase in organic synthesis

10.5 Concluding remarks

References

Chapter 11. Versatility of microbial laccases in industrial applications

Abstract

11.1 Introduction

11.2 Laccase-catalyzed organic synthesis

11.3 Biosensors and medical devices

11.4 Medical applications, healthcare, and cosmetics

11.5 Textiles and leather

11.6 Beverage and food

11.7 Limitations and future perspectives

Acknowledgments

References

Index

Copyright

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Dedication

Dedicated to our parents

List of contributors

Mayowa Agunbiade,     Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology, Bellville, South Africa

Arunima,     Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi, India

Fatemeh Aziziyan,     Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Nisha Bhardwaj,     Department of Chemical Engineering, Institute of Chemical Technology, Mumbai, Maharashtra, India

Sarita G. Bhat,     Department of Biotechnology, Cochin University of Science and Technology, Thrikkakara, Kochi, Kerala, India

Nicoletta Cascelli

Department of Chemical Sciences, University Federico II, Naples, Italy

Department of Organic and Inorganic Chemistry, University of Oviedo, Oviedo, Spain

Jesus D. Castilla-Marroquin,     Postgraduate College Campus Córdoba-Veracruz Km 348, Manuel Leon Congregation, Municipality of Amatlan of the Reyes, Veracruz, México

Bahareh Dabirmanesh,     Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Addolorata De Chiaro,     Biopox s.r.l., Naples, Italy

Farnoosh Farzam,     Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Georg M. Guebitz,     University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 20, BOKU-IFA, Tulln, Austria

Ricardo Hernández-Martínez,     CONAHCYT-Postgraduate College Campus Cordoba-Veracruz Km 348, Manuel Leon Congregation, Municipality of Amatlan of the Reyes, Veracruz, México

Francisco Hernández-Rosas,     Postgraduate College Campus Córdoba-Veracruz Km 348, Manuel Leon Congregation, Municipality of Amatlan of the Reyes, Veracruz, México

Jiya Jose,     Division of Microbiology, Department of Biosciences, Rajagiri College of Social Sciences, Kalamassery, Kochi, Kerala, India

Khosro Khajeh,     Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Khushi Khera,     Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi, India

Tukayi Kudanga,     Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Marilize Le Roes-Hill,     Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology, Bellville, South Africa

Vincenzo Lettera

Biopox s.r.l., Naples, Italy

Department of Chemistry and Chemical Technologie, University of Calabria, Arcavacata di Rende, Cosenza, Italy

Rekha Mehrotra,     Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi, India

Hamy Michel,     Department of Biotechnology, Cochin University of Science and Technology, Thrikkakara, Kochi, Kerala, India

Blessing Nemadziva,     Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Gibson S. Nyanhongo

University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 20, BOKU-IFA, Tulln, Austria

Biotechnology and Food Technology, University of Johannesburg, Johannesburg, South Africa

Roberto Parra-Saldivar,     Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico

Alaric Prins,     Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology, Bellville, South Africa

Virendra K. Rathod,     Department of Chemical Engineering, Institute of Chemical Technology, Mumbai, Maharashtra, India

Daniel Romero-Martínez,     Programa de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México

Shilja Sajeevan,     Department of Biotechnology, Cochin University of Science and Technology, Thrikkakara, Kochi, Kerala, India

Giovanni Sannia

Biopox s.r.l., Naples, Italy

Department of Chemical Sciences, University Federico II, Naples, Italy

Mauricio A. Trujillo-Roldán

Programa de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México

Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja CA, Mexico

Norma A. Valdez-Cruz

Programa de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México

Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja CA, Mexico

Preeti Verma,     Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi, India

Renate Weiss,     University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 20, BOKU-IFA, Tulln, Austria

Aarti Yadav,     Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi, India

Deepti Yadav,     Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Preface

Deepti Yadav and Tukayi Kudanga

Over the past three decades, laccases have generated interest due to their potential for industrial application. Although fungal laccases have traditionally been preferred due to their higher redox potential and broad substrate range, bacterial laccases have recently become the focus of attention based on their industrially relevant characteristics, such as activity over a broad pH range and in some cases, thermostability and resistance to common enzyme inhibitors. Despite these favorable characteristics, studies on bacterial laccases have lagged behind those of fungal enzymes until the last decade. While extensive literature is available on fungal laccases, there has not been a book dedicated to bacterial laccases.

This book provides a concise overview of bacterial laccases in a single volume, including foreseeable work in this area. Extensive studies have been conducted in a bid to fully exploit laccases for industrial applications. A brief historical timeline of the key contributions that have advanced knowledge in laccase research is provided, giving the reader the sequence of events from the discovery of the enzyme, its characterization, reaction mechanism, and structural elucidation to applications. An introductory chapter provides an overview of laccases, comparing fungal and bacterial laccases, their occurrence, function, structures, and coupling to mediator systems. The prospects and challenges of using bacterial laccases for industrial application are presented and set the scene for subsequent chapters that discuss advances made in response to these challenges. Among these challenges, there is the availability of large amounts of crude and purified enzyme. The second chapter focuses on the potential of agro-industrial wastes as feedstock for laccase production using solid-state and submerged fermentation, which potentially can scale up laccase production for cost-effective industrial use. While agro-industrial wastes provide substrates for cost-effective enzyme production, laccases from native sources offer an unfavorable combination of isozymes, limiting their usage due to low production levels and consequent high cost per unit of enzyme. Therefore a whole chapter has been dedicated to the recombinant expression of laccases in various hosts, such as Escherichia coli and Pichia pastoris, including the value of combining computational and experimental approaches for enhancing recombinant laccase production. Factors that affect different strategies for increasing recombinant enzyme production are also discussed.

The use of laccases has been limited in part because natural enzymes have not evolved to meet the requirements of complex industrial processes. Advances made in engineering laccases to match specific requirements are proffered; specifically, rational, semirational, and directed evolution approaches have been used in altering laccase properties. Using these engineering approaches, one can obtain robust biocatalysts not found in nature. Bioinformatic tools (e.g., homology modeling and molecular docking) used for the elucidation of structure-function relationships of the variant enzymes are discussed.

Immobilization is often used to reduce costs through recycling and reusing enzymes. Apart from increased stability and shelf life, immobilized enzymes are generally easier to handle and less sensitive to changes in reaction conditions such as pH variation. However, laccase activity can be compromised due to the reduced flexibility of immobilized enzymes and limited availability of substrate to the enzyme. Although a number of traditional supports are available for laccase immobilization, the search for cheaper, biodegradable, and thermostable materials that resist contamination and can be functionalized for efficient and effective immobilization, is continuing. Therefore the use of innovative materials such as waste materials from the agro-industrial sector, nanomaterials, magnetic and mesoporous materials, cross-linked enzyme aggregates, and biochar, as dynamic supports for enzyme immobilization, is attracting a lot of scientific interest. The wide range of support materials available means that immobilization must be tailored to the application to ensure that the intended goals are met. The facts and analysis offered in this book can greatly aid in determining the best matrix and method for laccase immobilization, not just for existing applications but also to allow for the development of novel applications.

Laccases oxidize a diverse range of substrates by electron abstraction to produce reactive radicals that couple to produce dimers, oligomers, or cross-coupling products, which is the basis of their application in organic synthesis. Cross-linking reactions are now widely applied in the food industry, for example, in dough strengthening and hydrogel formation. Enzymatic functionalization is a fast-growing branch of biotechnology which allows the adaptation of coupling reactions to graft functional compounds to materials such as wood or textiles, improving or altering their functional properties. Alternatively, radicals can couple to form insoluble polymers which can precipitate out of water bodies, effectively removing the compounds; this is particularly useful during bioremediation or clarification of drinks in the food industry. Radicals can also cleave covalent bonds, facilitating the degradation of xenobiotic compounds and lignin for biofuel or pulp and paper production. Oxidation–reduction reactions have been exploited in the biosensor industry. The wide substrate range of laccases, coupled with the subsequent diverse nonenzymatic reactions following laccase-catalyzed oxidation, has opened opportunities for the wide range of applications discussed in this book. Chapters have been dedicated to organic synthesis, material surface functionalization, delignification and detoxification processes, and bioremediation, while other applications have also been comprehensively reviewed. Though fungal laccases were traditionally used, bacterial laccase usage is gathering momentum due to their industrially relevant properties and advances in protein engineering which have facilitated the tailoring of their properties to industrial applications. Generally, bacterial laccases are more amenable to genetic manipulation than fungal laccases. Although laccases are generally considered versatile green catalysts (work with oxygen as cosubstrate and produce water as the only by-product), their application is often associated with many challenges, which are discussed in several chapters.

While this book is a good resource for solutions to challenges related to biotechnological applications of bacterial laccases, it is clear that the reflections of the authors converge at enzyme and substrate costs and suboptimal enzyme properties as the key challenges that will shape research direction in the next decade. It is therefore safe to assume that bacterial laccases will remain a topic of intense research to unlock their full potential as commercial enzymes.

The editors acknowledge all the authors for their expert contributions to this book, the reviewers for their constructive comments, and Blessing Nemadziva for assistance with the design of the book cover.

Chapter 1

Bacterial laccases: a general introduction

Tukayi Kudanga,    Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

Abstract

Laccases are oxidoreductases that oxidize a wide range of substrates, including phenols, diphenols, methoxy-substituted phenols, and alkyl and phenolic amines in a redox reaction that involves the concomitant reduction of molecular oxygen to water. Amongst the main producers of laccases are bacteria. Although they have been known for about three decades, laccases have not been fully exploited despite their potential, given that some are thermostable and are active over a broad pH range. This chapter introduces bacterial laccases and their potential and prospects for industrial application.

Keywords

Bacterial laccases; reaction mechanism; sources; applications

1.1 Introduction

Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are oxidoreductases that catalyze the abstraction of an electron from a suitable substrate (mainly phenolics, alkylamines, and phenolic amines) to the corresponding radicals. The radicals can then undergo a number of nonenzymatic reactions, including radical-mediated cleavage of covalent bonds, and homo- and hetero-coupling reactions to form dimers, trimers, other oligomers, polymers, and hybrid compounds. These radical-mediated processes have implications for many potential industrial applications. Oxidative coupling reactions are relevant in organic synthesis (Kudanga et al., 2017) or in bioremediation processes since polymeric materials precipitate and can be filtered out of aquatic bodies. Cleavage of covalent bonds, such as alkyl–aryl bonds, sometimes occurs in the presence of mediators, and can be applied in bioremediation to degrade xenobiotic compounds or complex polymers such as lignin. Although peroxidases can perform similar functions, they require a cosubstrate, hydrogen peroxide, which makes the process expensive and environmentally unfriendly.

Among laccases, fungal laccases have traditionally been the enzyme of choice, mainly because of their relatively higher activity compared to other laccases. However, fungal laccases are limited by the narrow pH range in which they are active and are susceptible to denaturing or inhibiting conditions often encountered during upscaling to less-controlled industrial applications. Unlike fungal laccases, bacterial laccases, in particular small laccases and those in spores of Bacillus, have attracted a lot of scientific and industrial interest as they are robust, thermostable (Yadav et al., 2018), active over a broad pH range (Dubé et al., 2008; Kudanga et al., 2009), and retain activity even in the presence of some putative inhibitors of fungal laccases and denaturing conditions (Machczynski et al., 2004).

Although bacterial laccases have become enzymes of interest, there are still a number of challenges that have limited their applications in industry. For example, low production yield by native microbial hosts is a major bottleneck for commercial applications. Most bacterial laccases are also produced intracellularly. Therefore, downstream processing steps are required which increase enzyme production costs. In order to exploit their full potential, it is imperative to understand the nature of bacterial laccases, their sources, the application challenges, and potential solutions to the challenges. This chapter introduces bacterial laccases, focussing on these key issues, and suggests possible solutions to challenges related to their application.

1.2 Laccase active site and reaction mechanism

Laccases are multicopper-containing enzymes that use a cluster of four copper atoms (namely Type 1 copper; Type 2 copper and two Type 3 copper atoms) to catalyze the oxidation of a suitable oxidizable substrate and simultaneously reduce molecular oxygen to water. Substrate oxidation takes place at the Type I copper atom. The abstracted electrons are then transferred to a trinuclear cluster (made up of the Type 2 copper atom and the two Type 3 copper atoms) where they reduce a molecule of oxygen to water, which is essentially a by-product of the oxidation process (Fig. 1.1).

Figure 1.1 Simplified structure of a laccase active site and the mechanism of laccase-catalyzed oxidation of substrate (sub) to its corresponding radical (sub.) ( Riva, 2006). Adapted from Riva, S., 2006. Laccases: blue enzymes for green chemistry. Trends Biotechnol. 24, 219–226.

The range of substrates oxidized by laccases is wide (Table 1.1) and the scope of substrates depends on the redox potential of the Type 1 copper site. The oxidation potential of a substrate largely depends on the difference in redox potential between the substrate and the enzyme Type 1 copper site. Essentially, for oxidation to occur, the enzyme should have a higher redox potential than the substrate. However, other factors such as the size, shape, and complexity of the substrate can also affect oxidation. In fungal laccases, the redox potential is usually high, up to E⁰=0.8 V (Piontek et al., 2002; Xu et al., 1998), while bacterial laccases have a lower redox potential of approximately 0.4–0.5 V (Durão et al., 2006; Gallaway et al., 2008; Kataoka et al., 2011). Therefore, bacterial laccases tend to have a narrower substrate range than fungal laccases.

Table 1.1

The substrate range of bacterial laccases can be widened by using laccase mediator systems (LMSs) (Fig. 1.2). In a LMS, the enzyme is used to oxidize an easily oxidizable substrate (with a low redox potential) to a corresponding radical. The radical is then used to oxidize a substrate of interest that is not readily oxidized by the enzyme because it has a particularly high redox potential or it is too large and complex to enter the enzyme active site. Consequently, a LMS expands the substrate range from simple oxidation of phenols, diphenols, and methoxy-substituted phenols to include nonconventional substrates such as adlerol (Uzan et al., 2010) and complex polymeric materials such as lignin (Kudanga and Le Roes-Hill, 2014).

Figure 1.2 Laccase mediator system for the oxidation of high redox potential or complex substrates.

1.3 Laccase sources

Laccases were first isolated from the Japanese lacquer tree Rhus vernicifera (Yoshida, 1883) but are now known to be ubiquitous in nature. They have been isolated in higher plants, prokaryotes, and insects, and are widespread in fungi (Kudanga et al., 2011). Although laccases have been known since the 19th century, bacterial laccases were only discovered over 100 years later, as explained in the next section.

1.3.1 Bacteria as sources of laccases

Although laccases are ubiquitous in fungi and most of the known and used laccases are of fungal origin, reports of laccases in bacteria have been increasing (Chauhan et al., 2017; Sharma et al., 2007). The first bacterial laccase was isolated from Azospirillum lipoferum, a bacterium that resides in the rhizosphere (Givaudan et al., 1993). Subsequently, records of the occurrence of prokaryotic laccases have been increasing with the enzyme frequently isolated in Bacillus spp. (Hullo et al., 2001) and several other Gram-positive bacteria such as Streptomyces, Geobacillus, Staphylococus, Aquisalibacillus, and Rhodococcus (Chauhan et al., 2017). However, laccases have been less frequently isolated in Gram-negative bacteria with Azospirillum, Psuedomonas, Stenotrophomonas, and Alteromonas as notable producers. The discovery of robust laccases, especially in Streptomyces and spores of Bacillus, has attracted a lot of industrial and research interest. The physiological role of Bacillus spores (which are naturally designed to withstand extreme physicochemical conditions) led to the speculation that spore laccases might have interesting industrially relevant properties. Subsequent characterization revealed that these laccases are indeed thermostable, showing activity at temperatures up to 85°C (Koschorreck et al., 2008; Martins et al., 2002) and are up to three times more resistant to putative laccase inhibitors thymine KCN, compared to fungal laccases (Kudanga et al., 2009). Similarly, the two-domain small laccase from some Streptomyces coelicolor strains is highly thermostable with a half-life of 10 hours at 80°C (Yadav et al., 2018) and over 7 hours at 90°C (Sherif et al., 2013). It is for this reason that small laccases and spore laccases have the greatest promise for industrial application.

1.4 Brief review of applications of laccases

Laccase oxidation results in the formation of reactive radicals, which can then undergo a number of nonenzymatic reactions that are relevant to the application of these enzymes in several industries. The radicals can couple to form dimers and oligomers, and this is the basis of the enzyme application in organic synthesis. Coupling of the radicals to form polymers has been applied for the removal of phenolic compounds in biofuel production and in bioremediation processes. Polymerization results in the formation of insoluble compounds that can then be filtered out of water bodies. In second-generation biofuel production, delignification processes produce phenolic compounds that must be removed to prevent the inhibition of subsequent enzymatic processes. Reactive radicals can also be used to cleave covalent bonds with application potential in the degradation of complex molecules. This has been the basis for delignification in the pulp and paper industry, in second-generation biofuel production, and in the degradation of xenobiotic compounds. Radicals can also facilitate oxidation-reduction reactions that have application potential in sensors and fuel cells. Table 1.2 summarizes the main applications of laccases and the basis for these applications.

Table 1.2

Although only a few of these applications have been piloted or commercialized, particularly in the pulp and paper and textile industries, laccases have shown real promise for application in many industries. In the wood industry, they have been used to produce binderless wood boards (Widsten and Kandelbauer, 2008). In the food industry, laccases have been used to remove phenolic compounds and stabilize beer and wines, and to crosslink and strengthen dough in the baking industry. Crosslinking reactions have also been applied in gelation to produce strong (Matiza Ruzengwe et al., 2023) and thermo-irreversible gels (Minussi et al., 2002; Norsker et al., 2000).

Perhaps the most widely studied area is the application of laccases in bioremediation, and a number of review articles have summarized the applications (Arregui et al., 2019; Kudanga et al., 2012; Strong and Claus, 2011). As pointed out earlier, these applications are based on polymerization reactions to facilitate the filtration of pollutants and oxidative degradation of compounds using radicals resulting from laccase-catalyzed oxidation reactions. These reactions are usually completed using LMSs. The laccase oxidizes small, easily oxidized (low redox potential) molecules to produce radicals, which in turn cleave covalent bonds of complex and high redox potential pollutants. Despite favorable properties of bacterial laccases such as robustness, activity over a broad pH range, and thermostability (especially small and spore laccases), most of these applications have used fungal laccases as they have a higher redox potential and a wider substrate range.

1.5 Prospects for bacterial laccases

Bacterial laccases have been the least utilized in laccase applications among the main laccase producers (fungi, plants, and bacteria), mainly because they have a low redox potential and therefore a low substrate range. Although LMSs could help solve this problem, this complicates reactions and adds costs to the process. In addition, most of the mediators are synthetic compounds that may be toxic and environmentally unfriendly, and therefore cannot be used for most applications, particularly in the food industry. Although there have been considerable advances in the search for natural mediators, they invariably add costs and in organic synthesis, additional purification steps are required to remove the mediator. Consequently, it is imperative to investigate approaches for tailoring bacterial laccases for industrial applications. Molecular approaches are now being widely investigated for the improvement of the properties of bacterial laccases. For example, Prins et al. (2015) used site-directed mutagenesis to improve the activity of the small laccase from S. coelicolor A3(2). Similarly, Gunne et al. (2014) showed that mutations near or distant to the Type 1 copper could increase redox potential and, in some cases, the resultant variants could oxidize high redox potential dyes. However, very often these mutations usually result in a small increase in redox potential, which means the enzymes are still inferior to fungal laccases in this respect. In addition, some modifications tend to compromise the thermostability properties of the enzymes (Prins et al., 2015).

Therefore, there is a need to explore other approaches to improve the application potential of bacterial laccases. More work can still be done on site-directed mutagenesis, and other protein engineering approaches are now being investigated. Typical examples include directed evolution by ancestral sequence reconstruction, computer-guided evolution, in vivo recombination of stable laccase chimeras using SCHEMA-RASPP, and consensus design. Although some promising results have been obtained using these approaches (Mateljak et al., 2020), there is still room for improvement. To improve applicability, these approaches can be combined with classical methods such as immobilization, use of cheaper substrates for enzyme production and catalysis, optimizing enzyme expression systems, and use of cheaper and eco-friendly mediators.

1.6 Conclusion

Bacteria are an important source of laccases. Although bacterial laccases have not been fully exploited for industrial application, research on their biochemical properties has shown industrially relevant properties such as thermostability and activity in extreme conditions that normally inhibit other laccases. However, more work is still required to improve the redox potential, optimize expression systems and reduce cost of the enzyme and substrates before the full potential of the bacterial laccases can be realized.

References

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Chapter 2

Agro-industrial wastes in bacterial laccase production by submerged and solid-state fermentation

Jesus D. Castilla-Marroquin¹, Francisco Hernández-Rosas¹ and Ricardo Hernández-Martínez²,    ¹Postgraduate College Campus Córdoba-Veracruz Km 348, Manuel Leon Congregation, Municipality of Amatlan of the Reyes, Veracruz, México,    ²CONAHCYT-Postgraduate College Campus Cordoba-Veracruz Km 348, Manuel Leon Congregation, Municipality of Amatlan of the Reyes, Veracruz, México

Abstract

During the last few years, laccase enzymes have generated interest due to their potential for industrial application in the biotechnology and environmental fields. Laccases have historically been produced by fungal strains; however, those produced by bacteria have shown unique characteristics such as stability at elevated temperatures and pH and are more amenable to genetic manipulation than fungal laccases. Bacterial strains can also produce enzymes intracellularly and extracellularly. Available lignin-rich agro-industrial wastes can be used as inducers to produce laccases efficiently and easily. There are numerous reports in the literature showing the production of laccases by bacteria using solid-state fermentation and submerged fermentation (SF). In this scenario, agro-industrial wastes are a potential feedstock that could be efficiently used to produce laccases by solid-state and SF using cost-saving processes. The most widely reported bacterial laccase producers in the literature are Rheinheimera, Lysinibacillus, Azospirillum, Bacillus, Geobacillus, Streptomyces, Rhodococcus, Staphylococcus, and Aquisalibacillus.

Keywords

Bacteria; laccase; submerged fermentation; solid-state fermentation; agro-industrial wastes

2.1 Introduction

Every year a large volume of waste is generated through the agricultural and food industries (Ravindran et al., 2018). The major problem regarding agro-industrial residues is that, in general, wastes are untreated, causing serious environmental problems as in most cases, agro-industrial wastes (AIW) are disposed of by burning, dumping, or landfilling (Sadh et al., 2018). Over the past decades, AIW have been studied as raw materials with an enormous potential to produce value-added products (Beltrán-Ramírez et al., 2019). AIW may have sustainable applications including the production of ethanol, essential oils, organic fertilizers, animal feeds, additives, and enzymes (Shirahigue and Ceccato-Antonini, 2020).

In recent years, laccase enzymes have been widely explored due to their potential for industrial application in several fields. Laccases have historically been produced by fungal strains, but studies of laccases produced by bacteria have shown that their unique characteristics make them very beneficial for industrial use. This chapter explores how laccases function, the potential for using AIW as feedstock for laccase production and two fermentation processes, solid-state and submerged fermentation (SF), which potentially can scale-up laccase production to the levels required for cost-effective industrial use.

2.2 Mode of action of laccases

Laccases are enzymes classified in the group of oxide-reductases (EC.1.10.3.2) and have gained much attention for their beneficial characteristics and because they are considered green and environmentally friendly. For example, a multicopper oxidase can catalyze single-electron oxidation in a wide range of organic substrates such as carbohydrates, aromatic and nonaromatic compounds, and phenolic and nonphenolic compounds which involve the reduction of oxygen to water (Liu et al., 2020; Senthivelan et al., 2016; Yang et al., 2020). When the substrate is a phenolic compound, a free radical is generated due to oxidation by laccase activity, resulting in an unbalanced product that may be subject to nonenzymatic reactions such as hydration, disproportionation, or polymerization, as shown in Fig. 2.1. On the other hand, when mediator compounds are present in the reaction medium, laccases show high oxidation capability which leads to the oxidation of nonphenolic lignin compounds, as shown in Fig. 2.2 (Patel et al., 2019).

Figure 2.1 Lignin compound nonphenolic subunits oxidation by laccase.

Figure 2.2 Lignin phenolic subunits oxidation by laccase.

Laccases are divided into two types—true laccases and false laccases—based on their activity in the presence of tyrosine. Those which show activity with tyrosine are identified as false laccases while those which do not show activity are identified as true laccases. They can also be differentiated based on three fundamental aspects, which classify them as blue or yellow-white, according to the following: blue laccases can be identified at 610 nm; blue laccases must have nonphenolic compounds as mediators for degradation; and blue laccases can only be produced in a liquid medium without any lignin, whereas yellow-white laccases can only be produced in a solid medium in the presence of lignin (Chauhan et al., 2017). Blue laccases are more easily produced and for this reason, they are more frequently