Chapter 2

Laccases: A Blue Enzyme for Greener

Alternative Technologies in the Detection
and Treatment of Emerging Pollutants

Melissa Rodríguez-Delgado and Nancy Ornelas-Soto

Abstract The continuous contamination of worldwide water bodies, by the pres-

ence of emerging pollutants, has raised great importance over the last decades. This
group of pollutants comprises a large variety of chemicals, comprehending house-
hold and personal care products, human and veterinary drugs, as well as industrial
compounds. Although, scientific data have made evident the potential threats of the
emerging pollutants to public and environmental health, there is still limited infor-
mation available concerning the ecotoxicity, concentration, and distribution of these
compounds, which makes their ecological regulation, detection, and treatment very
difficult. Thus, the search for green technologies to detect and treat potential envi-
ronmental pollutants is critical for ecological and human health protection. In this
context, laccases have gained scientific interest due to their broad substrate range,
including recalcitrant environmental pollutants, and their ability to use only oxygen
as a co-substrate. This work explores the potential of laccase enzyme as element of
biosensing and bioremediation, and identifies the drawbacks that have to be over-
come in order to demonstrate their feasibility and implement a large-scale process.

1 Introduction

To date, there is a critical environmental problem of contamination in water

resources with persistent, bioactive, and bioaccumulative substances, which cause
potential health and ecological effects (Petrovic 2003). Some of these chemicals are
daily life articles, such as household and personal care products, human and veteri-
nary drugs, which are extensively used and constantly released into the aquatic eco-
systems by human activities and direct discharges from wastewater treatment plants
(WWTPs) (Caliman and Gavrilescu 2009). These groups of substances are known

M. Rodríguez-Delgado (*) • N. Ornelas-Soto

Laboratorio de Nanotecnología Ambiental, Centro del Agua para América Latina y el Caribe,
Tecnológico de Monterrey, Monterrey, N.L. 64849, Mexico
e-mail: mmrodriguez@itesm.mx

© Springer International Publishing AG 2017 45

R. Singh, S. Kumar (eds.), Green Technologies and Environmental
Sustainability, DOI 10.1007/978-3-319-50654-8_2
46 M. Rodríguez-Delgado and N. Ornelas-Soto

as emerging pollutants and their detection and removal is crucial due to their high
persistence and toxicity, even at concentrations as low as ng/L (Bolong et al. 2009;
Gavrilescu et al. 2014). It is well-known that most of the emerging pollutants that
pass through conventional WWTPs are not completely removed (Caliman and
Gavrilescu 2009). Thus, the development of biosensors resulted as an effort in the
search for analytical tools capable to detect these kinds of contaminants at low con-
centrations with a high specificity (Rodriguez-Mozaz et al. 2006). However, once
the pollutants have been identified, a decontamination process has to be performed.
In this context, bioremediation of water resources by the use of biocatalysts, such as
enzymes, has been suggested in recent years. Enzymes are biomolecules that have
the ability to mediate reactions; severe conditions are not required for their action
and normally the by-products formed during the catalysis are benign, which implies
a great opportunity for the use of enzymes in bioremediation (Senthivelan et  al.
2016). Currently, several enzymes have been used in different biotechnological and
industrial applications; however, laccases enzymes have received special attention
due to its ability to oxidize a wide range of substrates, accompanying the reduction
of oxygen to water as a by-product of reaction (Rodríguez Couto and Toca Herrera
2006). Laccases are able to oxidize, polymerize, or transform diverse recalcitrant
substances into less toxic molecules; therefore, these enzymes could be suitable
biocatalysts for water bioremediation (Majeau et  al. 2010). However, several
drawbacks have to be surpassed in order to implement the use of laccases for pollu-
tion alleviation, mainly, because of the elevated expenses that a large-scale enzyme
production involves (Majeau et al. 2010).

2 Laccase

2.1 Laccase Source

As oxidoreductase enzymes, laccases possess the ability to oxidize diverse phenolic

compounds with the concomitant reduction of oxygen (Yaropolov et  al. 1994;
Morozova et  al. 2007a; Madhavi and Lele 2009). These biocatalysts have been
widely found as extracellular and intracellular enzymes in several organisms,
encompassing microorganisms (Bacillus), plants (genus Rhus) (Omura 1961), fungi
(such as genera Trametes, Cerrena, Ganoderma, Pycnoporus, and Coriolopsis)
(Chaubey and Malhotra 2002; Morozova et al. 2007b; Madhavi and Lele 2009), and
insects (M. sexta) (Bailey et al. 2004). In insect species, laccases are responsible for
the sclerotization process; whereas in Bacillus species, they play a role related to
pigmentation, pathogenesis, and the assembly of UV-resistant spores (Kramer et al.
2001; Claus 2003). In plants, laccases are found in the xylem and, together with
peroxidases, are involved in lignification (Omura 1961); moreover, fungal laccases
play a role in delignification (Singh Arora and Kumar Sharma 2010) and humifica-
tion processes (Morozova et al. 2007b).
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection… 47

2.2 Biocatalytic Mechanism and Applications

In an enzyme, the active site is the spot where the recognition, binding, and oxida-
tion of the substrate take place. The active site of laccases comprises four copper
nucleuses, each of them are grouped into three different classes of atoms according
to both their distribution in the enzyme (T1, T2, and T3 sites) and their spectro-
scopic nature (Piontek et al. 2002).
The T1 and T2 sites only possess one copper atom; the two remaining atoms are
found in the T3 site. Each type of copper has unique characteristics: type 1 is respon-
sible for the emblematic bluish color of the enzyme, is a hydrophobic cavity where
the substrate is oxidized during the catalytic mechanism of the laccase (Durán et al.
2002; Madhavi and Lele 2009); type 2 is colorless since no absorption in the visible
region is observed; meanwhile, type 3 exhibits a weak peak at 330 nm (Ba et al.
2013). The reduced form of T1 (resulted from substrate oxidation) donates an elec-
tron, which is sent to the T2/T3 trinuclear cluster (formed from T2 and T3 copper
atoms), which is where water formation occurs (reduction of oxygen) (Durán et al.
2002; Madhavi and Lele 2009) (Fig. 2.1).
The isoelectric point of laccases is around 4.0, showing their optimal perfor-
mance at acidic conditions, which has been related to the growth conditions where
the microorganisms produced these enzymes (Madhavi and Lele 2009). However, it
has been recently reported that laccases maintain high stability (above 60% of its
activity) at alkaline conditions (up to pH 8) (Ramírez-Cavazos et al. 2014b). On the
other hand, the thermostability, high redox potential, and the ability to oxidize a
wide range of substrate (including recalcitrant pollutants) are some other properties
that make laccase of particular interest to researchers (Giroud and Minteer 2013;
Ramírez-Cavazos et al. 2014b). Nowadays, the use of this enzyme is common in
some industries (mostly paper, food, and textile industries) (Morozova et al. 2007b).
However, the current trend is the use of laccase as biocatalysts in the bioremediation
of polluted waters by emerging pollutants (Almansa et al. 2004; Junghanns et al.
2005); in the generation of energy by bioelectrocatalysis in enzymatic fuel cells
(Meredith and Minteer 2012; Giroud and Minteer 2013; Holmberg et al. 2015); and
in the development of biosensors using this enzyme as bioreceptor, for food

Fig. 2.1 Model of the laccase active site and catalytic cycle
48 M. Rodríguez-Delgado and N. Ornelas-Soto

(Ghindilis and Yaropolov 1992; Gamella et al. 2006; Di Fusco et al. 2010), environ-
mental (Torrecilla et al. 2007; Tang et al. 2008), and medical applications (Quan and
Shin 2004; Ferreira et al. 2009).

2.3 Laccase Production in Agro-Industrial Residues

Despite the high potential of laccase for biotechnological and industrial purposes, it
is important to highlight that a large amount of enzyme is needed for a large-scale
process. According to Osma et al. (2011), the culture medium represents the highest
cost to the total expenses of laccase production; meanwhile, infrastructure costs are
the lowest. Thus, in order to overcome this issue, several studies have been focused
on (1) the development of stable genetic modifications of microorganism in order to
achieve the gene expression for laccase; (2) optimization and reduction of the costs
of culture media using agricultural wastes as cheap growth substrates (also helping
to alleviate the environmental pollution); (3) search for new strains of microorgan-
ism capable to produce laccase (Bhattacharya et  al. 2011; Yang et  al. 2012;
Theerachat et al. 2012; Nicolini et al. 2013; Ramírez-Cavazos et al. 2014a).
Ramírez-Cavazos et al. (2014a) tested a strain of Pycnoporus sanguineus, native
from northern Mexico, using a tomato juice as culture medium, resulting in laccase
titer of 143,000 U/L.  Fenice et  al. (2003) obtained a production of 4600 U/L in
olive-mill wastewater. Meanwhile, Songulashvili et al. (2007) reported the laccase
production using food wastes such as mandarin peelings, wheat bran and soy bran,
kiwi fruits, chicken feathers, and ethanol, obtaining a maximum laccase activity of
93,000–97,000 U/L, after the submerged fermentation of wheat bran and soy bran
by Ganoderma spp. Several agro-wastes such as mandarin and banana peel are sub-
strates rich in lignin carbohydrates and organic acids, which could act as inducers to
stimulate laccase production (Osma et al. 2007). In this context, several studies have
reported the use of solid supports such as grape seeds (Rodríguez Couto et al. 2006),
banana skin (Osma et  al. 2007), and groundnut (Couto and Sanromán 2006) as
growth substrates for fungi. However, the use of these complex substrates has as
drawback the subsequent use/purification of the laccase after fermentation; thus the
development of robust protocols that allow the use of crude enzymes (no purifica-
tion process) in biotechnological applications is required.

3 Laccase-Based Biosensor for Detection of Emerging

Pollutants

3.1 Emerging Pollutants in Water Reservoirs

Over the past two decades, the constant presence of emerging pollutants in world-
wide water supplies has gained great relevance. This type of pollutants includes a
large variety of chemicals used in daily life such as household compounds, personal
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection… 49

care products, and drugs for human and animal uses (Daughton and Ternes 1999).
Although scientific data have made evident the potential threats of the emerging
pollutants to public and ecological health, there is still information that has not been
assessed (Horvat et al. 2012) concerning the environmental toxicity, concentration,
distribution, and transformation of these compounds in water bodies, which makes
their ecological regulation, detection, and treatment very difficult (Deblonde et al.
2011). These pollutants are typically released into the environment via anthropo-
genic activities such as agriculture practices, industrial, and human discharges
(Murray and Ormeci 2012). The emerging pollutants are commonly classified as
endocrine disruptors, pharmaceutical compounds, and personal care products
(Daughton and Ternes 1999).
The endocrine disruptors are compounds that mimetize the action of hormones
in the organisms, causing the alteration of the endocrine system, which has been
related to sexual disorders, cancer, and even chronic diseases (Caliman and
Gavrilescu 2009; Rezg et  al. 2013). Nowadays, the presence of chemicals that
exhibit hormone alterations and are involved in the elaboration of plastics and
household products has been widely reported, e.g., surfactants, flame retardants,
parabens, and plasticizers (Rodriguez-Mozaz et al. 2004). Meanwhile, the chemi-
cals present in personal care products and considered as emerging pollutants encom-
pass disinfectants (triclosan), conservation agents, fragrances (celestolide, tonalide,
galaxolide), and UV screens (octyl-dimethyl-PABA, octyl-methoxycinnamte,
homosalate) (Caliman and Gavrilescu 2009). On the other hand, the pharmaceutical
compounds, as emerging pollutants, encompass human and veterinary drugs that
have been widely found in water supplies such as antibiotics, nonsteroidal anti-
inflammatory drugs, and beta-blockers (Deblonde et al. 2011). Numerous studies
have reported the presence of personal care products, pharmaceutical compounds,
and endocrine disruptors in several water supplies at alarming concentrations
(Meisenheimer et al. 2002; Mompelat et al. 2009; Einsiedl et al. 2010; Lapworth
et  al. 2012). Teijon et  al. (2010) reported a monitoring survey of pharmaceutical
compounds in the water sampled from a WWTP and from the aquifer that is
recharged by the discharge of this WWTP, resulting in the detection of these chemi-
cals at concentrations of μg/L.
A large number of analytical protocols have been developed for the detection of
these kind of pollutants, mainly by chromatography and spectroscopy techniques,
since these methods are extremely accurate and capable to detect very low concen-
trations; however, they require complicated pretreatment sample, trained personnel,
high operating costs, and they lack on-site applicability (Teijon et al. 2010). In this
context, the need for portable analytical instruments, capable to field monitoring
with high selectivity, sensitivity, and short assay times, has promoted the design of
new devices such as biosensors (Marco and Barceló 1996).
A biosensor is an instrument capable of measuring a specific target molecule in
a sample, taking advantage of its affinity toward a specific bio-element of recogni-
tion (bioreceptors, e.g., immunoreagents, enzymes) (Dzyadevych et al. 2008). There
are three essential elements that comprise a biosensor (Fig. 2.2). The bioreceptor
interacts specifically with the analyte present in the sample, producing a biochemical
50 M. Rodríguez-Delgado and N. Ornelas-Soto

Fig. 2.2 Essential components of a biosensor: bioreceptor, electrochemical transducer, electric

pathway in the production of the biosensor response and typical electrical response of the laccase-
based electrode biosensors

reaction derived from this interaction, which is amplified and converted into quan-
tifiable electrical signals (Dzyadevych et  al. 2008). The transducer is in intimate
contact with the bioreceptor and is responsible for the translation of the biochemical
event into an electric signal (Dzyadevych et al. 2008).
The use of enzymes as bioreceptors has been widely employed in biosensing due
to their stability and easier control of their recognition properties in comparison
with other biomolecules such as antibodies or cells (Rogers 2006). In this context,
the oxidase enzymes (e.g., tyrosinase, peroxidase, and laccase) appear as good can-
didates due to their ability to catalyze reactions where electron transference occurs,
which can be used as the transduction principle. Laccase enzymes have some ben-
efits over the other oxidases, which make them highly interesting for biosensing
applications; their thermostability, versatility to react with a wide range of sub-
strates, no cofactors are needed to perform the catalysis and the formation of water
as by-product are some of these advantages (Munteanu et al. 1998).

3.2 Immobilization Methods

In general, an efficient protocol of immobilization is developed to facilitate the bio-

molecule recovery and reusability. Thus, the immobilization of the bioreceptor
would prolong the life of the biosensor and assure its work stability (Liu et al. 2006).
The method of immobilization to apply must provide the best conditions to assure
the highest stability of the bioreceptor and this will depend on the inherent proper-
ties of the recognition element; the operational requirements of the measurement,
the target molecule, and the transduction principle are also important factors to take
into account in the design of a biosensor (Singh et al. 2008). The immobilization
methods most commonly employed for laccase attachment are covalent coupling,
adsorption, cross-linking, encapsulation, and entrapment (Fig.  2.3) (Fernández-
Fernández et al. 2013).
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection… 51

a O b c
HOOC O NH
H+O
H+O

SH
SH
SH

d e NH2
NH2 NH2

NH2
NH2

Fig. 2.3 Immobilization methods employed in biosensors. (a) Covalent coupling, (b) Adsorption,
(c) Cross-linking, (d) Encapsulation, (e) Entrapment

The covalent coupling requires the chemical reaction between the functional
groups in the carrier with the biomolecule (mainly, through the amino acid residues
in their structure) forming covalent bonds; however, it is important to avoid the
binding of the enzyme through amino acids within the active site, since that would
compromise the biocatalyst activity, resulting in their inhibition (Arroyo 1998). The
immobilization via adsorption takes places by weak interactions between the bio-
molecules and the solid support, e.g., Van der Waals interactions or ionic forces;
despite the simplicity and inexpensive requirements of the protocol, this scheme
presents the disadvantage of an unsteady fixation of the bioreceptors under pH
changes, modification of the polarity or the ionic strength alterations, resulting in
the leakage of the molecules (Brady and Jordaan 2009).
The cross-linking involves the generation of intramolecular links within the mol-
ecules of the enzyme (Arroyo 1998). Cross-linked enzyme crystals present high
catalytic activities and operational stability; however, high quantities of highly puri-
fied enzyme are required; under this scheme, it is important to maintain the pH and
salt concentration stable (Bryjak et al. 2007). Encapsulation refers to the confine-
ment of the biomolecule within semipermeable spheres made of polymers (Rochefort
and Kouisni 2008); meanwhile, entrapment is based on the retention of the enzyme
within a polymeric grid; this method assures the integrity of the enzyme structure,
however it presents diffusion problems and constant loss of the biomolecules due to
differences in the size of the pore in the grid (Ibarra-Escutia et al. 2010).

3.3 Transduction Principles

3.3.1 Electrochemical Transducers in Environmental Applications

Electrochemical transducers refer to the element in a biosensor that translates the

transference of electrons occurring during the reaction of an enzyme (immobilized
on an electrode) with a substrate (Marco and Barceló 1996; Thévenot et al. 2001).
52 M. Rodríguez-Delgado and N. Ornelas-Soto

Laccase enzymes possess mechanical properties and electron transference abilities

that make them excellent bioreceptors in electrochemical biosensors (Table 2.1).
The electrochemical transducers in biosensors are able to measure conductome-
try, potentiometry, and voltammetry principles (Thévenot et  al. 2001).
Conductometric methods monitor the development of a redox reaction through
changes in the conductivity of an electrolytic solution, as a response of the charged
products formed by the interaction of the bioreceptor with the analyte (Ronkainen
et al. 2010). Potentiometric biosensors measure the changes of potential due to a
biochemical reaction occurring between two electrodes (either, reference or indica-
tor electrodes) (Thévenot et al. 2001). Voltammetric measurements are based on the
changes of the current during a biochemical reaction on a working electrode; mean-
while, the voltage applied is varied (Chawla et al. 2012). Amperometry is a classifi-
cation of voltammetry; under this scheme, the potential is maintained constant
during the reaction measurement (Thévenot et al. 2001).
Chen et al. (2015) reported the immobilization of laccase in a nanocomposite of
gold nanoparticles by a cross-linking method, achieving a direct electron transfer
that resulted in a highly sensitive biosensor . Meanwhile, Das et al. (2014) immobi-
lized a Trametes versicolor laccase in a nanocomposite matrix comprising of
osmium tetroxide on poly 4-vinylpyridine, multiwalled carbon nanotubes; obtain-
ing a current response against pyrocatechol, a genotoxic and mutagenic phenol, in
the concentration range of 3.98–16.71 nM and a limit of detection of 2.82 nM with
a sensitivity of 3.82 ± 0.31 nA/nM. Vianello et al. (2004) immobilized a Rigidoporus
lignosus laccase onto a gold carrier obtaining a detection limit of 0.5 mg/L. Tang
et  al. (2006) reported the immobilization of an hybrid bioreceptor, consisting of
horseradish peroxidase and laccase enzymes for the detection of E. coli density
through the redox reaction of polyphenols generated by the metabolism of the E.
coli. On the other hand, the development of new nanomaterials has improved the
electrochemical analysis performed by biosensors. In this context, Mei et al. (2015)
prepared a mixture of palladium and copper to cover a support of graphene oxide in
order to build a platform for laccase immobilization; obtaining a detection limit of
2.0 mM for catechol. According to Rodríguez-Delgado et al. (2015), amperometric
methods are highly employed in laccase-based biosensors; meanwhile, conducto-
metric and potentiometric methods are not very often employed for this use.

3.3.2 Optical Transducers in Environmental Applications

Optical absorption and fluorescence emission measurements have been extensively

used to characterize colored or chromophoric compounds. Based on this concept,
the spectroscopic properties of by-products formed by laccase catalysis are
employed for the detection of target molecules in optical biosensors (Zoppellaro
et al. 2001). Laccase has the ability to produce a reddish dye when catalyzes the
coupling oxidation of phenols in presence of 3-methyl-2 benzothiazolinonehydra-
zone (MBTH) (Setti et  al. 1999). Based on this reaction, Abdullah et  al. (2007)
reported the detection of catechol at concentrations as low as 0.33 mM.  The
Table 2.1 Electrochemical biosensors using laccase as bioreceptor for detection of pollutants
Laccase Immobilization Working
sources method electrode Target molecule Sensing parameters Matrix References
Coriolus Covalent Gold Catechol Dynamic range Synthetic (Gupta
hirsutus coupling 1–400 μM et al. 2003)
Sensitivity
15 μA/mM
Cerrena Electrodeposition Platinum Hydroquinone Dynamic range Synthetic (Jędrychowska
unicolor 2.0–60 μM et al. 2014)
Sensitivity
2.34 ± 0.11 μA/mM
Not reported Entrapment Nafion/laccase Catechol Dynamic range Real samples (Chen
glassy carbon 0–7 μM et al. 2015)
Trametes Entrapment Platinum Phenol Dynamic range Wastewater (Timur
versicolor 0.40–6.0 μM et al. 2004)
Catechol 0.20–1.0 μM
l-DOPA 2.0–20 μM
Aspergillus Phenol Dynamic range
niger 0.40–4.0 μM
Catechol 0.4–15 μM
l-DOPA 0.4–6.0 μM
Coriolus Covalent Indium Polyphenolic
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection…

1.6 × 103 to E. coli (Tang

versicolor coupling tin oxide compounds of 1.0 × 107 cells/mL solutions et al. 2006)
E. coli metabolism
53

(continued)
 54

Table 2.1 (continued)

Laccase Immobilization Working
sources method electrode Target molecule Sensing parameters Matrix References
Pleurotus Electrostatic Indium Phenol Limit of detection Wastewater (Kushwah
ostreatus attachment tin oxide 0.5–4.5 μM et al. 2011)
Catechol 0.4–15 μM
Coriolus Adsorption Graphite Catechol Limit of detection Synthetic (Yaropolov
hirsutus hydroquinone 2 μM et al. 1995)
Coriolus Entrapment Glassy Catechol Dynamic range Synthetic (Liu
versicolor carbon/chitosan 1.2–30 μM et al. 2006)
Limit of detection
0.66 μM
Rigidoporus Covalent Gold 1,4-Hydroquinone Sensitivity 3 nA/μM Olive oil (Vianello
lignosus coupling wastewater et al. 2004)
Trametes Entrapment Glassy-carbon/ Hydroquinone Dynamic range Synthetic (Yaropolov
hirsute Cetyl ethyl 0.1–3.0 μM et al. 2005)
poly(ethyleneimine)/
Nafion
Trametes Adsorption Graphite Catechol Dynamic range Synthetic (Portaccio
versicolor and covalently up to 0.1 mM et al. 2006)
coupling Sensitivity
196.0 μA/mM
Trametes Entrapment Gold Catechol Dynamic range Synthetic (Xu
versicolor 0.67–15.75 μM et al. 2010)
Pleurotus Entrapment Glassy carbon Phenol Dynamic range Synthetic (Kushwah and
ostreatus 0.5–4.5 μM Bhadauria
M. Rodríguez-Delgado and N. Ornelas-Soto

2010)
Laccase Immobilization Working
sources method electrode Target molecule Sensing parameters Matrix References
Ganoderma Covalent Gold with cooper Polyphenol/ Dynamic range Synthetic (Chawla
sp. Rckk02 coupling nanoparticles- guaiacol 1–500 μM et al. 2011)
carboxylated Sensitivity
multiwalled 0.694 μA μM−1 cm−2
carbon nanotubes-
polyaniline
Trametes Entrapment Glassy carbon Pyrocatechol Dynamic range Real samples (Das et al.
versicolor 3.98–16.71 nM 2014)
Sensitivity
3.82 ± 0.31 nA/nM
Cerrena Electrolytic Platinum o-Amino phenol Not reported Real samples (Cabaj et al.
unicolor deposition catechol phenol 2011)
Cerrena Entrapment Platinum 4-tertbutylcatechol Dynamic range Not reported (Kochana et al.
unicolor 2–89 μM 2008)
4-methylcatechol 0.21–15 μM
3-chlorophenol 0.98–7.9 μM
Catechol 0.2–23 μM
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection…
55
56 M. Rodríguez-Delgado and N. Ornelas-Soto

detection was made by spectrophotometric measurements of the reddish compound

formed from the coupling of MBTH films (stacked in nafion silicate) to radicals
quinone and/or phenoxy produced by laccase enzymatic oxidation (Abdullah et al.
2007). Sanz et al. (2012) studied the reaction of phenol catalyzed by Trametes ver-
sicolor laccase, using a polyacrylamide film sensor immobilized with the enzyme,
obtaining a limit of detection of 0.109 mM.
Surface plasmon resonance (SPR) phenomenon is also an optical principle
employed for biosensing, since almost two decades ago. SPR measurements are
performed at a fixed angle of incidence and the monitoring is done by detecting
changes in the amount of the reflected light, which is correlated to changes in mass
on the surface due to binding events (between the bioreceptor and the analyte)
(Estevez et al. 2012). In this context, Surwase et al. (2016) reported a first attempt
for the detection of phenolic compounds in water by using laccase enzyme as bio-
receptor in a SPR device.

4 Laccase as Biocatalyst for Removal of Emerging Pollutants

Several studies have reported the presence of endocrine disruptors, personal care
products, and pharmaceutical compounds in diverse water bodies at important con-
centrations (Snyder et al. 2003; Caliman and Gavrilescu 2009). Thus, the presence
of these pollutants in water supplies has become an important issue in terms of treat-
ment technologies for water cleaning, mainly because of the highly resistance of
these compounds to common removal techniques. Advanced oxidation techniques
based on UV/ozone exposure have obtained efficient yields of removal/inactivation
(Esplugas et al. 2007); however, these processes are expensive (Lloret et al. 2012)
and in some cases the by-products generated are more toxic than the parent pollut-
ant (Sein et  al. 2008). Therefore, laccases appear as strong biocatalysts to be
employed in bioremediation treatments, since can react under mild conditions with
a broad substrates range, encompassing recalcitrant pollutants, and generate non-
harmful by-products. Fukuda et al. (2004) demonstrated the removal of the endo-
crine activity of the by-products from bisphenol A; catalyzed by Trametes villosa
laccase (Fukuda et  al. 2004). Likewise, Cabana et  al. (2007a) reported nonestro-
genic activity in the products of reaction by the catalysis of bisphenol A, triclosan,
and nonylphenol by employing Coriolopsis polyzona laccase.
The removal of emerging pollutants from water supplies using laccase could be
achieved under several schemes: (1) free enzyme; (2) immobilized enzyme; and (3)
cells from culture broths.
Several studies have reported the use of free laccases for emerging pollutants
removal. Yang et al. (2013) reported 27% of diclofenac elimination. The total elimi-
nation of estrone, 17β-estradiol, estriol, and 17α-ethinylestradiol (Auriol et al. 2006,
2007, 2008), and oxybenzone (Garcia et al. 2011) was achieved by Trametes versi-
color laccase. Almost complete removal of 2,4-diclorophenol was observed by free
laccase at acidic pH (Jia et al. 2012), temperatures around 30 and 50 °C (Zhang
2 Laccases: A Blue Enzyme for Greener Alternative Technologies in the Detection… 57

et al. 2008; Gaitan et al. 2011; Qin et al. 2012; Xu et al. 2013), and using elevated
concentrations of enzyme (Zhang et al. 2008). Jia et al. (2012) investigated the deg-
radation of 2,4-dichlorophenol by a photocatalytic–enzymatic treatment, achieving
90% within 2 h with the coupled degradation process.
The use of mediators is also common during enzymatic degradations; these com-
pounds are small-size molecules that can extend the ability of an enzyme to react
toward noncommon substrates; also are stable and reusable by various cycles
(Majeau et al. 2010). ABTS, 1-hydroxy-benzotriazole (HBT), nitroso-2-naphthol-
2,6-disulfonic acid (NNDS), Syringaldehyde, 4-Acetylamino-TEMPO 4-hydroxy-
TEMPO, Violuric acid (VIO), and p-Coumaric acid are some of the mediators most
widely employed for laccase catalysis (Majeau et al. 2010). Ji et al. (2016) reported
the elimination of carbamazepine using as mediators p-coumaric acid, syringalde-
hyde, and acetosyringone, obtaining 60% of degradation after 96 h. Meanwhile,
Margot et al. (2015) assessed the potential of laccase to remove sulfamethoxazole
and isoproturon with three mediators: ABTS, syringaldehyde, and acetosyringone,
showing complete transformation within a few hours. Almost total biotransforma-
tion of diclofenac by laccase was also obtained using 1-hydroxybenzotriazole
(Nguyen et al. 2013), syringaldehyde, and violuric acid (VA) (Lloret et al. 2010,
2013).
In terms of immobilization, Krastanov (2000) studied the degradation of
β-naphtol, observing a complete removal after a hybrid treatment with laccase from
Pyricularia oryzae and tyrosinase. Lante et al. (2000) immobilized P. oryzae lac-
case on a polyethersulfone membrane, obtaining 18% of β-naphtol removal. Le
et al. (2016) provided a novel immobilization technique for laccase on copper algi-
nate for real wastewater treatment, showing 89.6% of triclosan removal after 8 h
treatment. Nguyen et al. (2014) reported an enzymatic membrane reactor for the
degradation of bisphenol A and diclofenac, obtaining >85% and >60% removal,
respectively, by laccase from Aspergillus oryzae. Meanwhile, Chen et  al. (2016)
immobilized laccases on the surface of yeast cells for treatment of bisphenol A
(46% removal after 6 h) and sulfamethoxazole (47% removal after 30 h of treat-
ment). Nevertheless, there are just a few works that address the degradation of
emerging pollutants in a real matrix and under real reaction conditions (pH, tem-
perature, ionic strength); this is important to consider since some matrix compo-
nents could decrease the laccase activity and therefore decrease the degradation
yield. Rodríguez-Delgado et al. (2016) tested the biotransformation of the micro-
pollutants: diclofenac, 5,7-diiodo-8-hydroxyquinoline, β-naphtol, and
2,4-diclorophenol using laccase from P. sanguineus CS43 in groundwater samples,
observing a reduced bioconversion for β-naphtol and 2,4-diclorophenol in the real
samples in comparison with the synthetic buffer matrix. Biotransformation of
bisphenol A, 4-nonylphenol, 17-α-ethynylestradiol, and triclosan were tested in
groundwater, as well (Garcia-Morales et al. 2015).
The presence of some ions in a reaction matrix has been reported to induce struc-
tural modifications in the active site of the enzyme (Zilly et al. 2011). For instance,
halide anions have been related to the interference on the transference of electrons
within the active site of the laccase enzyme (Enaud et al. 2011; Margot et al. 2013);
58 M. Rodríguez-Delgado and N. Ornelas-Soto

meanwhile, cyanide and calcium provoke the separation of the copper atoms from
the enzyme (Cabana et al. 2007b). Kim and Nicell (2006) observed that bisphenol
A biodegradation was adversely affected by nitrite, thiosulfate, and cyanide.

5 Future Perspectives and Conclusions

The use of laccases as bioreceptors in biosensors promotes the development of new

technologies for environmental monitoring in terms of pollutants screening.
However, the immobilization of the bioreceptor is a crucial step in biosensors
design, thus more research has to be focused on (1) the creation of new materials for
support purposes that diminish the loss of the enzymes once they are immobilized;
(2) the improvement of the current immobilization methods to assure an oriented
binding of the enzyme in order to safeguard the integrity of the active site, resulting
in the recovery of the enzymatic activity once the immobilization was performed;
and (3) the use of genetic modifications to extend the laccase stability under difficult
catalysis conditions, assuring the recycling of the bioreceptor and thus the lifetime
of the biosensor.
The exploitation of laccase enzymes as element of bioremediation opens up
enormous possibilities in terms of future treatment technologies for water cleaning.
However, unlike biosensor applications, an elevated amount of enzyme is required
for bioremediation. Therefore, one of the crucial issues that avoid large-scale use of
the laccase is related to the high cost of enzyme production; thus further research
should be focused on diminishing the cost that represents the growth mediums for
the microorganisms during the laccase production. Furthermore, future research
needs to look at (1) the test of the interaction of hybrid treatments in bioremediation,
e.g., photocatalytic–enzymatic, enzymatic–enzymatic, whole cell–enzymatic, etc.,
(2) the study of the structure and toxicity of the by-products formed by laccases
catalysis under different reaction conditions, and (3) the implementation a pilot-
scale process, where the laccase treatments would be performed under real reaction
conditions (pH, temperature, and matrix composition), which consider possible
matrix interactions over the catalysis.

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