Heterologous laccase production and its role in industrial applications

Abstract

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry.

Introduction

Laccase (benzenediol:oxygen oxidoreductase; p-diphenol oxidase EC 1.10.3.2), a blue multi copper oxidase (MCO), was first discovered by Yoshida (1883)¹ in the latex of the Chinese or Japanese lacquer trees (Rhus sp.). In plant these enzymes seem to be involved in lignification, in wound healing as part of an herbivore or pathogen defense response, and in iron metabolism.² Fungal laccases were discovered during the 19^th century.³ Laccases are thought to be nearly ubiquitous among fungi, and their presence has been documented in virtually every fungus examined so far.⁴ In fungi, laccases carry out a variety of physiological roles including morphogenesis, fungal plant-pathogen/host interaction, stress defence and lignin degradation.⁴ In general, fungal laccases are monomeric globular proteins of approximately 60–70 kDa with acidic isoelectric point (pI) around pH 4.0, although several exceptions exist. The majority of fungal laccases are extracellular enzymes generally glycosylated, with an extent of glycosylation usually ranging between 10 and 25% and only in few cases higher than 30%. Multiplicity of laccase genes is a common feature in fungi and plants, and the production of several laccase isoenzymes has been observed in many species. Some phenoloxidases showing “laccase properties” have been purified from larval and adult cuticles of several insects, probably associated with the sclerotization process.⁵ Now, there is increasing evidence for the existence of proteins with typical features of the multi-copper oxidase enzyme family also in prokaryotes.⁶ Laccase encoding genes have been found in gram-negative and gram-positive bacteria, including species living in extreme habitats. Early reports of laccases in actinomycetes were based on rather non-specific substrate reactions, but have been verified for some bacteria of genera Streptomyces.⁷ Role attributes to bacterial laccases include copper homeostasis, sporulation, or pigmentation of spores to confer resistance to stress factors such as UV radiation or hydrogen peroxide.⁸

Laccases couple the four single electron oxidations of a reducing substrate to the four electron reductive cleavage of the dioxygen bond, using four Cu atoms distributed into three sites, defined according to their spectroscopic properties.⁴ Typical metal content of laccases includes one type-1 copper (T1), one type-2 (T2) and two type-3 copper (T3) ions, with T2 and T3 arranged in a trinuclear cluster (TNC). The type 1 site contains the blue copper, whose tight coordination to a cysteine is responsible for an intense SCys → Cu(II) charge transfer transition at around 600 nm, giving the typical blue color to the enzyme. The T2 shows a characteristic electron paramagnetic resonance (EPR) spectrum, clearly distinct from that of T1, whereas T3 coppers are anti-ferromagnetically coupled and EPR-silent ions.

T1 exhibits a planar triangular coordination with the sulfur atom of a cysteine and with the Nδ1 nitrogen of two histidines. The three T2/T3 ions are arranged in a triangular fashion and coordinated to a strongly conserved pattern of four His-X-His motifs.⁹ Six of such histidine residues coordinate the T3 copper pair, whereas the T2 is coordinated by the remaining two histidine residues. Electrons from the reducing substrate are extracted from the T1, the primary electron acceptor, and then transferred to the TNC through a highly conserved His-Cys-His tripeptide, where the four electron reduction of dioxygen to water takes place.

A number of 3D structures of laccases have been solved. All the fungal laccases exhibit a similar molecular architecture organized in three sequentially arranged cupredoxin-like domains.⁴ The T1 is located in domain 3, whilst the TNC cluster is embedded between domains 1 and 3 with both domains providing residues for coppers coordination.

Laccases exhibit an extraordinary range of natural substrates (phenols, polyphenols, anilines, aryl diamines, methoxy-substituted phenols, hydroxyindols, benzenethiols, inorganic/organic metal compounds and many others) which is the major reason of their attractiveness for dozens of biotechnological applications.¹⁰ The repertory of laccase catalyzed oxidative reactions can be enlarged by the means of the so-called mediators. Suitable redox mediators are good laccase substrates, whose oxidized forms have a half-life long enough to permit their diffusion towards otherwise non-oxidable laccase substrates (non-phenolic substrates or large molecules), and possess high oxidation potential to allow laccases to indirectly oxidize them.¹¹

A number of applications for laccases have been proposed in several industrial sectors, such as textile, food, paper and pulp, pharmaceutical, chemistry, nano-biotech, cosmetic, along with their application in bioremediation. As a fact, laccases can be adopted (1) to bleach textiles;¹² (2) to eliminate undesirable phenolics responsible for the browning, haze formation and turbidity development in clear fruit juice, beer and wine;¹³ (3) to bleach wood pulp;¹⁴ (4) to synthesize various functional organic compounds such as drug, dyes;^15,16 (5) to produce various polymers;¹⁷ (6) to detect molecules in biosensor devices;¹⁸ (7) to produce power in biofuel cells;¹⁹ (8) and to dye hair.²⁰ As far as bioremediation is concerning, laccases may be applied to decolorize textile effluents;²¹ to degrade plastic waste having olefin units;²⁰ to eliminate odor emitted from places such as garbage disposal sites, livestock farms, or pulp mills;²⁰ to remove phenolic compounds from olive oil mills^20,22 and pulp mills wastewaters;²⁰ and to decontaminate soils from polycyclic aromatic hydrocarbon (PAH).²⁰

Laccase Recombinant Expression

Laccase production from native sources cannot meet the increasing market demand due to low yields incompatibility of the standard industrial fermentation processes with the conditions required for the growth of many microorganisms. Recombinant protein expression in easily cultivable and handling hosts can allow higher productivity in shorter time and reduces the costs of production. The versatility and scaling-up possibilities of the recombinant protein production opened up new commercial opportunities for their industrial uses.²³ Enzyme productivity can be increased by the use of multiple gene copies, strong promoters and efficient signal sequences, properly designed to address proteins to the extracellular medium, thus simplifying downstream processing. Moreover, protein production from pathogenic or toxin-producing species can take advantage of safer or even GRAS (generally recognized as safe) microbial hosts. In addition, protein engineering can be employed to improve the stability, activity and/or specificity of an enzyme, thus tailor made enzymes can be produced to suit the requirement of the users or of the process. Enzymes of superior quality have been obtained by site-directed or random mutagenesis, where single changes in aminoacidic sequences yield improvement in biochemical (pH optimum, thermostability, substrate specificity) and catalytic parameters (v_max, K_M and K_i). Also, pooling and recombining parts of similar genes from different species or strains by “DNA shuffling” methods, yields remarkable improvements in enzymes in a very short amount of time.²⁴

Since the beginning of nineties, recombinant expression of laccases has been field of research and matter of debate of many researcher groups. Heterologous expression of laccases in bacteria, yeasts, filamentous fungi and plants has been reported, along with examples of homologous expression.^25–30

Laccase heterologous expression in Escherichia coli has been often used as a strategy to get around the problem of obtaining laccases not easily producible in natural hosts. The recombinant expression of Bacillus subtilis CotA in E. coli has allowed its deep characterization, structure solving, and functional evolution.^8,31,32 However, very often the production yield is low, and recombinant enzymes form aggregates difficult to purify.³³ On the other hand, recombinant production of Streptomyces coelicolor laccase (SLAC) in Streptomyces lividans has yielded considerable large amount of laccase (350 mg l⁻¹) with high purity.³⁴

The laccase from the ligninolytic fungus Cyathus bulleri has been just recently expressed in E. coli making it the first fungal laccase to be expressed in a bacterial host.³⁵ A collection of references on recombinant expression of laccases in bacterial hosts is available in Table 1.^36–46

Table 1.

List of heterologously expressed laccases in bacteria References regarding laccase engineering are in red.

Laccase	Source	Bacteria	Reference
PpoA	Marinomonas mediterranea		36
			8
			31
CotA	Bacillus subtilis		37
			38
			39
			32
EpoA	Streptomyces griseus		40
STSL	Streptomyces lavendulae REN-7	Escherichia coli	33
Lbh1	Bacillus halodurans		41
SLAC	Streptomyces coelicolor		42
Tth-laccase	Thermus thermophilus HB27		43
McoA	Aquifex aeolicus		44
CotA	Bacillus licheniformis		45
			46
SilA	Streptomyces ipomea		7
Cbu-laccase	Cyathus bulleri		35
SLAC	Streptomyces coelicolor	Streptomyces lividans	34

References regarding laccase engineering are in red.

Plants have been successfully used as hosts for the recombinant expression of fungal and plant laccases. Expression of secreted recombinant laccases can result, for example, in phytoremediation systems.^47,48 Furthermore, the overexpression of a potato laccase (PPO) in tomato conferred to the transgenic plant an enhanced resistance against a bacterial pathogen.⁴⁹ A collection of references on laccase recombinant expression in plants is available in Table 2.^50–54

Laccase recombinant expression has also been accomplished in insect Sf9 cells, via a baculovirus expression system, leading to a deep characterization of the insect laccase from tobacco hornworm, Manduca sexta.⁵⁵

A deeply review of the available literature data on recombinant expression and engineering of laccases in yeasts and filamentous fungi is given in the following paragraphs.

Yeast Recombinant Systems

Yeasts offer the quickness of microbial growth and ease of microbial and gene manipulation of bacteria along with the ability to perform eukaryote-specific post-translational modifications, such as proteolytic processing, disulfide bridge formation, and glycosylation. Yeast growths are economical, usually give high yields, and are low demanding in terms of time and effort.⁵⁶ Furthermore, yeasts are organisms suitable for creating new enzymes with desirable characteristics. Saccharmoyces cerevisiae and Pichia pastoris have often been the yeasts of choice for laccase recombinant expression. Also plant laccases were produced in these two yeasts.^47,57 As a fact, P. pastoris is always a better producer of recombinant laccases with respect to S. cerevisiae, giving yields from 8⁵⁸ to 17 mg l⁻¹.⁵⁹ Hyper-glycosylated laccases are often produced in such conventional yeasts. First results of heterologous expression of fungal laccases in the non-conventional yeasts Kluyveromyces lactis,⁶⁰ Yarrowia lipolytica,⁶¹ and Pichia methanolica⁶² have become available in 2005, with yields comparable to those obtained using other yeasts and a limited extent of glycosylation. A complete list of heterologously expressed laccases in yeasts is reported in Table 3.^63–117

Table 3.

List of heterologously expressed laccases in yeasts

Laccase	Source	Yeast	Reference
PO1	Coriolus hirsutus		63
PO2	Coriolus hirsutus		63
LCC1	Trametes versicolor		64
LCC2	Trametes versicolor		64
			65
LAC2	Pinus taeda		57
LCC1	Trametes sanguinea M85-2		66
			67
			68
MtL	Myceliophthora thermophyla		69
			70
			71
			72
LACIII	Trametes versicolor		73
LAC1	Melanocarpus albomyces		74
			75
LAC1	Trametes sp. strain C30		76
LAC2	Trametes sp. strain C30	Saccharomyces cerevisiae	76
LAC3			76
			77
LAC3/LAC1	Trametes sp. strain C30		78
LAC3/LAC2			78
LAC3/LAC5			78
POXC	Pleurotus ostreatus		60
			60
			79
POXA1b	Pleurotus ostreatus		80
			81
			82
LCC1	Pycnoporus coccineus		83
LCCα	Trametes versicolor UAMH8272		84
LAC2	Coprinellus congregatus		85
Ery3	Pleurotus eryngii		86
POX3	Pleurotus ostreatus		87
POX4	Pleurotus ostreatus		87
			88
LCC1	Trametes versicolor		89
			90
LAC1	Cryptococcus neoformans		91
LCCI	Trametes versicolor		92
LAC1	Pycnoporus cinnabarinus		58
LCC1	Trametes sanguinea M85-2		66
LCCIV	Trametes versicolor		93
LAC4	Pleurotus sajor-caju		94
LCCT	Panus ridis		95
LCC	Fome lignosus		96
			97
LAC1	Gossypium arboreum	Pichia pastoris	47
LAC1	Flammulina velutipes		98
LCCPol	Pleurotus ostreatus		99
LCC1	Trametes versicolor		100
LCC2	Trametes versicolor		100
LCC1	Trametes trogii		59
LAC a	Trametes sp. AH28-2		101
LACB	Trametes sp. AH28-2		102
			103
LACD	Trametes sp. 420		104
			105
			106
LACC	Trametes sp. 420		107
GLlac1	Ganoderma lucidum		108
LAC	Pycnoporus sanguineus		109
LCC2	Trametes trogii		110
POXC	Pleurotus ostreatus
POXA1b	Pleurotus ostreatus		60
LCC1	Trametes trogii	Kluyveromyces lactis	111
			112
POXA3b	Pleurotus ostreatus		113
POX3	Pleurotus ostreatus		87
POX4	Pleurotus ostreatus		87
LACIIIb	Trametes versicolor	Yarrowia lipolytica	61
			114
LAC1	Pycnoporus cinnabarinus		115
LCC1	Trametes versicolor	Pichia methalonica	62
			116
			117

References regarding laccase engineering are in red.

It is worth to note the considerable variability of production yields with respect both to cDNAs expressed and to the host used for the heterologous expression. As a fact, LCC1 laccase from Trametes versicolor is actively expressed in P. pastoris⁸⁸ and not in S. cerevisiae.⁶⁴ Also POXC and POXA1b laccases form Pleurotus ostreatus have been expressed at higher level in K. lactis than in S. cerevisiae,⁶⁰ whereas POX3 from the same source displayed the opposite behavior.⁸⁷ Moreover, when K. lactis has been used as host, it is possible to note different level of expression between cDNAs originating from the same organism.⁶⁰ Codon preferences between the host and the fungal source seem not to explain such differences. The inability of yeasts to process different laccases post-translationally with the same efficiency may influence the observed “selectivity” in expression.⁷⁶

Laccase expression in yeasts has been investigated taking into account an array of different parameters with controversial results. For instance, both native laccase or yeast signal peptides (S. cerevisiae α-factor or invertase; K. lactis killer toxin; Y. lipolytica XPR2) have been used with different results for various expressed enzymes. Therefore, the best performing signal peptide to drive the secretion of recombinant laccases in yeasts seems not to be a priori predictable. The effect of pH, temperature, media composition, inducers, and copper has also been evaluated. High copper concentration is required for the production of active laccase at a post-translational step in a heterologous host.⁸³ All together, these variable yields hinder forecast of the most suitable host, or the most promising laccase to express.

The availability of recombinant yeast systems has allowed to address some targets. For instance, the production of fuel ethanol from renewable raw materials has been obtained using laccase-expressing S. cerevisiae with an increased resistance of the yeast to phenolic inhibitors due to lignocellulose hydrolysates.⁶⁵ Experiments to ascribe a pathogenic function to laccase from Criptococcus neoformans have been conducted using recombinant enzymes.⁹¹ Moreover, the availability of the recombinant expression system of the P. ostreatus heterodimeric laccase POXA3 in K. lactis allowed inferring a role to the quaternary structure in stabilizing laccase.¹¹³ Furthermore, heterologous expression has been often used as a strategy to get around the problem of obtaining laccase isoforms not easily producible in natural hosts.^{66,76,84,86,87,93}

Some other authors have reported the production of recombinant laccases as a model (1) to better understand copper trafficking and the hierarchy of copper distribution in the cell;⁷³ (2) to examine the protein quality control mechanism;⁸³ (3) to study the induction of its own promoter by oxidative stress;⁸⁵ (4) to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeasts.¹¹¹

Development of applications of recombinant laccases from yeasts is discussed in a next section.

Filamentous Fungi Recombinant Systems

The attraction of filamentous fungi as production hosts is based on their natural ability to secrete large amounts of proteins into the growth medium.¹¹⁸ Until now, only a limited number of fungal host species has been explored for recombinant protein production. Filamentous fungi that dominate the markets as production hosts are the asexually reproducing Aspergillus niger, Aspergillus oryzae and Trichoderma reesei. Consequently, most reports on heterologous laccase expression concern studies on these fungi, and few examples in the genetically well-characterized Aspergillus nidulans, and in the patented expression system of Aspergillus sojae. A complete list of heterologously expressed laccases in filamentous fungi is reported in Table 4.^119–158 The yields of heterologous laccases obtained in filamentous fungi are considerably higher than those obtained in yeasts, generally in the range of hundreds of milligrams per litre. The production levels reported for the expression of Melanocarpus albomyces laccase in T. reesei are the highest heterologous laccase expression levels reported so far, allowing the obtainment of 230 mg l⁻¹ in shake-flask cultures, 290 mg l⁻¹ in batch fermentations, and 920 mg l⁻¹ in fed-batch fermentation.¹²¹ Actually, a series of investigations on laccases has been accomplished through heterologous production in fungi. The conclusions inferred by Xu and co-workers¹²⁸ regarding redox potential, substrate specificity and stability of recombinant laccases expressed in A. oryzae well illustrate this point. Likewise, the recombinant laccase of Trametes villosa in A. oryzae was investigated to elucidate the reaction mechanism of the reduction of dioxygen to water by stopped-flow experiments and under steady-state conditions.¹³⁴ Moreover, the availability of high yields of recombinant proteins has allowed solving 3D structures of the laccase from Coprinus cinereus¹³⁹ and bacterial laccase from S. coelicolor expressed in A. oryzae,¹⁴⁴ and that from M. albomyces expressed in T. reesei.^123,126

Table 4.

List of heterologously expressed laccases in filamentous fungi. References regarding laccase engineering are in red.

Laccase	Source	Filamentous fungus	Reference
PrL	Phlebia radiata		119
Gene IV	Trametes versicolor		120
			121
			122
		Trichoderma reesei	123
LAC1	Melanocarpus albomyces		124
			125
			126
			75
Laccase	Stachybotrys chartarum		29
LCC1	Rhizoctonia solani		127
LCC2	Rhizoctonia solani		127
			127
LCC4	Rhizoctonia solani		128
			129
			130
			131
			128
			132
LCC1	Trametes villosa (Polyporus pinsitus or Coriolus pinsitus)		133
			18
			130
			134
			135
			136
			128
			129
MtL	Myceliophthora thermophyla	Aspergillus oryzae	18
			130
			137
			135
			138
LCC1	Coprinus cinereus		139
			130
			135
	Scytalidium thermophilum		140
			128
LAC1	Pycnoporus cinnabarinus		141
LCC1	Pycnoporus coccineus		83
LAC1	Trametes versicolor UAMH 8272		142
LAC4	Trametes versicolor UAMH 8272		142
LCC1	Lentinula edodes		143
LCC4	Lentinula edodes		143
SLAC	Streptomyces coelicolor		144
LAC	Schizophyllum commune	Aspergillus sojae	145
			146
LAC1	Pycnoporus cinnabarinus		147
			148
Oxidase B	Stachybotrys chartarum		149
			150
			151
LCS-1	Ceriporiopsis subvermispora		152
LCC1	Trametes versicolor	Aspergillus niger	153
			154
Gene IV	Trametes versicolor		155
pox2	Pleurotus ostreatus		156
Laccase	Stachybotrys chartarum		29
Pel3	Pleurotus eryngii		157
LCC1	Trametes versicolor
LCC2	Trametes versicolor		100
			158
LCS-1	Ceriporiopsis subvermispora	Aspergillus nidulans	152
Laccase	Stachybotrys chartarum		29

References regarding laccase engineering are in red.

Laccase heterologous expression systems have also been employed as model to measure the effectiveness of different promoters,²⁹ besides other more specific aims, such as (i) to study the effect of constitutive activation of the Unfolded-protein response (UPR) pathway on the production of heterologous protein in A. niger;¹⁵³ (ii) to examine the protein quality control mechanism;⁸³ (iii) to select strains with higher production levels of heterologous proteins;¹⁵⁶ (iv) to evaluate the physiological state of an organism in biotechnical processes;^124,125 (v) to establish the effects of reduced protease activity on the stabilities of secreted recombinant proteins.¹⁵⁴

Development of applications of recombinant laccases from filamentous fungi is discussed in a next section.

Laccase Engineering

The availability of established recombinant expression systems for laccase isoenzymes has allowed their engineering with the aim of deepening knowledge of structure function relationships and of improving several enzymatic features for specific industrial needs. As a fact, (rational or random) mutagenesis has often been used to generate laccase variants, either in yeasts and in filamentous fungi. In their pioneering work, Xu and co-workers^129,132 reported significant changes in pH optimum, K_M and k_cat for mutated fungal laccases. New insights into the binding of the reducing substrate into the active T1 site of a laccase produced by T. versicolor have been provided by site directed mutagenesis along with induced modifications in catalytic properties of the enzyme.¹¹⁴

The role of the C-terminus in basidiomycetous and ascomycetous laccases has been evaluated using site directed and random mutagenesis. Gelo-Pujic and co-workers⁹² found that the barrier to heterogeneous electron transfer is reduced when the C-terminus of LCCI from T. versicolor is truncated. An additional consequence of truncating the C-terminus of LCCI is a shift in the reduction potential of the active site to a lower value. When Kiiskinen and Saloheimo⁷⁴ studied the expression of M. albomyces in S. cerevisiae, they found that the introduction of a stop codon after the native processing site at the C-terminus gives rise to a sixfold increase of laccase activity. A role for the C-terminal tail of P. ostreatus POXA1b in affecting both catalytic performance and stability properties of the enzyme has been inferred by Autore and colleagues.⁸² More recently, results obtained with M. albomyces laccase clearly confirmed the critical role of the last amino acids in its C-terminus.⁷⁵ The deletion of the last four amino acids dramatically affected the activity of the enzyme. Moreover, the crystal structure of the mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the TNC geometry. Further insights in the significance of the laccase C-terminal tail have also been provided through random mutagenesis. Functional expression of a laccase from Myceliophthora thermophila by directed evolution has been first reported by Bulter and co-workers⁶⁷ giving a 170-fold increase of total activity, thus leading to the highest production yet reported for a laccase in a yeast (18 mg l⁻¹). A 22-fold increase in k_cat has also been observed. The most effective mutation (10-fold increase in total activity) adjusts the protein sequence to the different protease specificities of the heterologous host, thus confirming the role played by C-terminus processing in acquiring functional structures. A P. ostreatus POXA1b has undergone directed evolution through random mutagenesis, and one of the selected mutant has been found mutated in a variable and mobile loop at the C-terminus.⁷⁹ Molecular dynamic simulations on 3D model structure of this mutant have shown the mutation affects flexibility of some regions of the protein, thus leading to an improved stability and activity of the enzyme.

Other techniques to randomly mutate laccases have been employed. Directed evolution followed by saturation mutagenesis rendered laccases able to tolerate high concentration of organic cosolvents.^68–72 A higher laccase production and an increased thermal stability have been obtained in P. pastoris through mutagenesis with low-energy nitrogen ion implantation.¹⁰¹ Random mutagenesis through ethyl methane sulfonate-based (EMS) technique has improved laccase production up to 144 mg l⁻¹ in P. pastoris.⁹⁷

Taking inspiration from evolutionary pathways within the blue copper binding domain (BCBD) protein family, laccase chimeras through yeast mediated homologous recombination of Trametes sp. strain C30 laccase cDNAs have been constructed.⁷⁸ The catalytic efficiency of the best-performing hybrid (LAC131) is 12-fold higher than that of the parental enzyme (LAC3). Compared to studies involving mutagenesis, this increase is one of the highest ever observed in a single mutational step, thus confirming how homologous recombination constitute a valuable tool set to study the plasticity of the enzyme.

Recombinant Laccases as Tools for Greening Industry

Laccases have shown a great potential within a variety of industrial applications, where they represent an attractive route for “greening” chemical processes. Owing to the heterogeneous properties observed among laccases from various sources,¹⁵⁹ an ever-increasing suite of native laccases has been applied to different biotechnological processes, with the aim to find the most suitable enzyme for a specific application.^160,161 However, only few examples of industrial uses of laccases currently exist. The major obstacle to their practical use is the large amount of enzymes required to meet industrial targets. Important breakthroughs towards an industrial use of laccases have been made by their recombinant expression in optimized hosts and production of genetically modified tailored biocatalysts.¹⁶² It is worth noting that the first industrial laccase preparation (DeniLite^®), launched by Novozyme in 1996 for denim finishing, is based on a recombinant and thermostable M. thermophila laccase expressed in A. oryzae.¹³⁶

Most of biotechnological applications with recombinant laccases are based on the same commercial preparation. DeniLite^® has been applied to assemble amine-derivatized platinum electrodes for phenol detection. This sensor has shown a very fast response and a remarkable long-term stability towards p-phenylenediamine,¹⁶³ catechol and catecholamines, with submicromolar detection limits.¹⁶⁴ The same laccase preparation has been employed in the construction of stable and high-sensitive ionic liquids-based biosensors, for the detection of rosmarinic acid in plant extracts,¹⁶⁵ rutin¹⁶⁶ and luteolin,¹⁶⁷ resulting in a low cost, reproducible and stable analytical method. Similarly, Kulys and co-workers¹⁸ developed graphite- or printed graphite-electrode based biosensors for environmental surveillance of phenolic compounds, by covalent immobilization of two recombinant fungal laccases from Polyporus pinsitus (T. villosa, Coriolus pinsitus) and M. thermophila, commercially available from Novozymes. Since the balance between optimal pHs for laccase function and substrate reversibility has been shown to be responsible for pH profile of catechol and catecholamines biosensors,¹⁶⁴ it can be expected that modifications of enzyme properties by recombinant expression of mutated variants may result in fine-tuning of biosensor performances.

Recombinant laccases have been widely applied in bioremediation purposes, especially for the treatment of synthetic dyes^59,80,109 and, more recently, of toxic polychlorinated biphenyls (PCBs),¹⁴² and PAHs.¹⁶¹ The purified recombinant Lcc1 from Trametes trogii, expressed in P. pastoris differently decolorizes several synthetic textile dyes, depending on their chemical structures. The extent of decolorisation is enhanced by the addition of synthetic mediators. Moreover, this enzyme has proved to be stable and active in the presence of moderate amounts of organic solvents.⁵⁹ A potential use of a recombinant Pycnoporus sanguineus laccase expressed in P. pastoris for the treatment of dye-containing effluents has also been suggested by Lu and co-workers in light of its remarkable ability to degrade, at different extents, four synthetic dyes belonging to different classes (azo, anthraquinone, triphenylmethane and indigo).¹⁰⁹ Finally, a POXA1b laccase mutant, selected for its improved stability in a wider pH range, has been successfully applied to dye decolorisation. This variant shows a further enlargement of dye degradation ability with respect to the wild-type, being also able to decolorize a recalcitrant dye with a complex stilbene type structure.⁸⁰

In view of an application to real colored wastewaters, laccase treatment of a synthetic dye house effluent, containing various reactive dyestuffs and auxiliary chemicals, has been studied in a batch reactor using the commercial preparation DeniLite^®. Significant correction of some water quality parameters (color, BOD, TOC, COD and toxicity) has been achieved and a reliable kinetic model has been developed to simulate the decolorisation process.¹⁶⁸

As far as PCBs degradation is concerned, two laccase isoenzymes from T. versicolor produced in A. oryzae are effective towards all tested hydroxyl-PCBs, with higher chlorinated hydroxy-PCBs (HO-PCB) being less susceptible to laccase treatment than lower chlorinated HO-PCBs. Interestingly, these isoforms show different specificities in oxidation of HO-PCB congeners.¹⁴² In a similar report, four T. versicolor laccase isoenzymes, expressed in P. pastoris, exhibit different efficiencies towards PAHs oxidation.¹⁶¹

Laccases have also found interesting applications in biopulping and biobleaching of lignocellulosic materials for paper manufacturing.^141,148 Sigoillot and co-workers investigated the pulp bleaching efficiency of P. cinnabarinus laccase expressed in two distinct Aspergilli hosts, in comparison with the native enzyme. The results obtained, together with the observed differences in redox potentials of the recombinant laccases, have been ascribed to the host-specific processing.¹⁴¹ In order to improve laccase treatments of pulp, Ravalson and co-workers synthesized a chimeric laccase by fusing P. cinnabarinus laccase lac1 to the carbohydrate binding module (CBM) of A. niger cellobiohydrolase B.¹⁴⁸ The chimeric protein was investigated for its softwood kraft pulp biobleaching potential in comparison with the native counterpart. By conferring to the chimeric protein the ability to bind to a cellulosic substrate, CBM addition greatly improves laccase delignification properties. In a similar approach, laccases for bleaching carotenoid-containing stains on fabrics, have been engineered. Peptide sequences, selected for their ability to specifically bind to carotenoid stains, have been linked to C-terminus of Stachybotrys chartarum laccase. The targeted peptide-laccase fusion demonstrated enhanced catalytic properties on stained fabrics.¹⁵¹

Fungal laccases are ideal green catalysts for many transformations in organic synthesis, spanning from oxidation of functional groups and coupling of phenols and steroids, to construction of carbon-nitrogen bonds and synthesis of complex polymers.¹⁰ In an interesting example, a recombinant laccase from M. thermophyla, supplied by Novozymes A/S, has been applied to the synthesis of a resveratrol dimer, a compound exhibiting promising antioxidant activity. The reaction has been carried out on a preparative scale, in very mild conditions resulting in improved yields in comparison with the analogous chemically catalyzed reaction.¹⁵

Improved tolerance to high concentrations of organic solvents is an enviable quality for laccase application in organic chemistry, since most of the transformations are carried out at high concentrations of organic solvents in which laccases may undergo unfolding, thereby losing their activity.¹⁰ Zumarraga et al. addressed this target by selecting an enzyme able to tolerate high concentrations of cosolvents⁷⁰ after five rounds of directed evolution of a laccase from M. thermophila. Regarding immobilization, the recombinant expression of an histidine-tagged Trametes sp.strain C30 laccase has allowed the oriented binding of a fully active monolayer of laccases on a chemically modified gold electrode. Such an immobilization strategy may be useful to modulate the electrical communication between an electrode and a redox protein site bond on its surface, as well as to improve ligand detection in solution.⁷⁷

Biocatalytical production of elemental iodine (I₂)—an attractive antimicrobial molecule—by oxidation of iodide, has been investigated using the recombinant laccases from P. pinsitus (rPpL), M. thermophila (rMtL), C. cinereus (rCcL), and Rhizoctonia solani (rRsL) in presence of methyl syringate as mediator. Tested enzymes show different kinetic behavior during the reaction. As a fact, the fitting kinetic data have revealed that the reversibility of the reaction increases for laccases with lower redox potential copper type I.¹³⁰

Recombinant laccases have also been employed as important bio-control measures to safe-guard or improve the quality and acceptability of food and beverages. For example, a T. versicolor UAMH 8272 laccase, has been successfully used for eliminating the highly toxic and mutagenic toxin AflatoxinB (AFB1) in food sources. The degradation has also shown to coincide with a significant and typical dose response loss of mutagenicity of the AFB1 molecule.¹⁵⁸ In another interesting report, a spectrophotometric method has been developed for antioxidant activity determination in “rich with antioxidant” food samples, by using P. pinsitus and M. thermophila laccases expressed in A. oryzae. The method, based on simultaneous oxidation of the antioxidant and an highly reactive laccase substrate producing chromophoric radical cation, allows the detection of submicromolar concentration of an antioxidant.¹³⁵

This review surveys the recent research on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry (Fig. 1). Enhanced protein production, and genetic tailoring of the enzyme profiles have been carried out successfully to construct novel recombinant enzymes for industry.

Figure 1.

Schematic representation of the steps towards “greening” chemical industry.

When producing recombinant biocatalyst to address a specific industrial need, the choice of expression host strain cannot be made solely on the basis of production yields. Other aspects, such as regulatory issues, play a very important role in this choice. Moreover, patents and intellectual property rights call for searching for expression hosts other than the species traditionally used.

Note

Dedicated to the memory of our missed friend and colleague Sophie Vanhulle who died suddenly and tragically.

Table 2.

List of heterologously expressed laccases in plants

Laccase	Source	Plant	Reference
LCC1	Trametes versicolor	Zea Mays	50
Ltlacc2.1-4	Liriodendron tulipifera		51
Ltlacc2.2		Nicotiana tabacum L. Cv. BY2 cells	2
LCC1	Lentinula edodes		52
LCC1/LCC4			53
LacIII	Coriolus versicolor	Nicotiana tabacum	48
MalL	Melanocarpus albomyces	Oryza sativa	54
PPO	Solanum tuberosum	Lycopersicon esculentum	49
LAC1	Gossypium arboretum	Arabidopsis thaliana	47

References regarding laccase engineering are in red.