Journal of Environmental Management 309 (2022) 114676

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Catalytic roles, immobilization and management of recalcitrant

environmental pollutants by laccases: Significance in sustainable
green chemistry
Syeda Fauzia Farheen Zofair a, Sumbul Ahmad a, Md. Amiruddin Hashmi a,
Shaheer Hasan Khan a, Masood Alam Khan b, Hina Younus a, *
a
Enzymology Laboratory, Interdisciplinary Biotechnology Unit, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India
b
Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah, 51452, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Keywords: We are facing a high risk of exposure to emerging contaminants and increasing environmental pollution with the
Laccase concomitant growth of industries. Persistence of these pollutants is a major concern to the ecosystem. Laccases,
Immobilization also known as “green catalysts” are multi-copper oxidases which offers an eco-friendly solution for the degra­
Bioremediation
dation of these hazardous pollutants to less or non-toxic compounds. Although various other biological methods
Laccase-mediator system
exist for the treatment of pollutants, the fact that laccases catalyze the oxidation of broad range of substrates in
Green chemistry
the presence of molecular oxygen without any additional cofactor and releases water as the by-product makes
them exceptional. They have a good possibility of utilization in various industries, especially for the purpose of
bioremediation. Besides this, they have also been used in medical/health care, food industry, bio-bleaching, wine
stabilization, organic synthesis and biosensors. This review covers the catalytic behaviour of laccases, their
immobilization strategies, potential applications in bioremediation of recalcitrant environmental pollutants and
their engineering. It provides a comprehensive summary of most factors to consider while working with laccases
in an industrial setting. It compares the benefits and drawbacks of the current techniques. Immobilization and
mediators, two of the most significant aspects in working with laccases, have been meticulously discussed.

1. Laccases: occurrence and properties
Laccase was first described by Yoshida in 1883 from the exudates of
Japanese lacquer tree Rhus vernicifera, from which the designation lac­
case was derived (Yoshida, 1883). In 1896, it was also discovered in
fungi by Bertrand (1896). Laccases are not only restricted to plants and
fungi, but they are also present in bacteria and insects (Alexandre and
Zhulin, 2000; Dittmer et al., 2004). Fungal origin laccases plays an
important role in plant pathogenesis, morphogenesis, stress defence,
fungal plant-pathogen/host interaction, pigment production and
degradation of lignocellulosic material (Alcalde, 2007; Liu et al., 2017;
Martina et al., 2018). While plant laccases participate in the radical
based mechanisms of lignin polymer formation, in insects they play
main role in cuticle sclerotization by catalyzing catechol in their cuticles
to corresponding quinines (Arakane et al., 2005; Baldrian, 2006). Lac­
cases have a molecular mass ranging from 33 to 130 kDa as reported by
various researchers (Driouichit et al., 1992; Guo et al., 2017). Molecular
weight of monomeric fungal laccase is around 50–110 kDa, and it has an
isoelectric point around pH 4. More than 100 laccases have been puri­
fied till date from fungi, most of which are from white rot

Abbreviations: LMS, laccase-mediator system; RMF, rotating magnetic field; 2, 6-DMP; 2, 6-Dimethoxyphenol; ABTS, 2,2′ -azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid); HBT, 1-hydroxybenzotriazole; HAT, hydrogen atom transfer; VA, violuric acid; TEMPO, 2,2’,6,6’; tetramethylpiperidine-N-oxyl, AA; acetylacetone,
CNT; carbon-nanotubes, PEI; polyethylene imine, HNT; halloysite nanotubes, A-M-HNTs-GTA; aminopropyltriethoxysilane-magnetic-Halloysite nanotubes-Glutar­
aldehyde, 2,4-DNP; 2, 4-Dinitrophenol; EPA, Environmental Protection Agency; BPA, bisphenol A; PAHs, polyaromatic hydrocarbons; EDCs, endocrine-disrupting
chemicals; ACE, active crude extract; SSF, solid-state fermentation; BOD, biological oxygen demand; COD, chemical oxygen demand; CPC, controlled porosity carrier;
WRF-MFC, white-rot fungi microbial fuel cell; OMW, olive mill waste; HPI, N-Hydroxyphthalimide; NHA, N-hydroxyacetanilidine; CSM, combinatorial saturation
mutagenesis; CueO, copper efflux oxidase; PCR, polymerase chain reaction.
* Corresponding author.
E-mail address: hyounus.cb@amu.ac.in (H. Younus).

https://doi.org/10.1016/j.jenvman.2022.114676
Received 6 September 2021; Received in revised form 8 January 2022; Accepted 2 February 2022
Available online 9 February 2022
0301-4797/© 2022 Elsevier Ltd. All rights reserved.
S.F.F. Zofair et al.
Fig. 1. Copper centres of laccase from Trametes versicolor (adapted from Piontek et al., 2002). Measurements are in Armstrong (1 Å = 10−
basidiomycetes. Most of these white rot fungi produce more than one
isoenzyme encoded by multiple structural genes (Blaich and Esser,
1975). Most fungal laccases are monomers, however some of them
exhibit homodimeric or heterodimeric structure (Min et al., 2001; Pal­
mieri et al., 2003). Despite the fact that laccase was discovered in 1883,
it attracted researchers only after their potential in enzymatic degra­
dation of wood by white rot fungi was recognized. Since then, several
laccases have been studied extensively with respect to their structure,
function, substrate specificity, copper binding site, industrial applica­
tion, bioremediation and green synthesis.
Laccases (E.C.1.10.3.2., para-bezenediol:dioxygen oxidoreductases,
p-diphenol oxidase) are blue multi-copper oxidases containing four
copper atom per monomer. Majority of the known laccases typically
contains four copper ions of three types. These T1, T2 and T3 sites can be
identified on the basis of their spectroscopic properties. T1 or type 1
copper, is called ‘blue’ copper and displays an intense absorption band
around 600 nm, detectable by electron paramagnetic resonance (EPR);
T2 shows very weak absorption band in the UV–Vis region, detectable in
EPR spectra; and a binuclear centre T3 displays an absorption band at
330 nm, not detectable in EPR spectra due to anti-ferromagnetic
coupling mediated by a bridging hydroxide ligand (Manole et al.,
2008; Solomon et al., 1996). T2 and T3 form a tri-nuclear centre (Fig. 1).
T1 copper bonds with two histidine and one cysteine residue (Manole
et al., 2008). Cysteine residue is replaced by phenylalanine or leucine in
fungal laccases. The linkage of copper and sulphur of cysteine residue
present in the catalytic site is responsible for “blue” colour of the
enzyme. Similar enzymes which lack this T1 Cu atom are called “yellow”
or “white” laccases (Piontek et al., 2002). Substrate oxidation takes
place at T1 because of its high redox potential and easy accessibility to
the substrate in comparison to the tri-nuclear cluster T2/T3. The
resulting electrons are transferred through a strongly conserved
His-Cys-His tripeptide motif to T2 and T3 site (Messerschmidt, 1997). T2
copper atom has 2 histidine residues while, both T3 Cu has total of 6
2
Journal of Environmental Management 309 (2022) 114676
10
m).
histidine residues (Manole et al., 2008). These residues help in electron
transfer from the substrate to the tri-nuclear structure where four elec­
tron reduction of molecular oxygen to water takes place with simulta­
neous oxidation of substrates to corresponding free radical which
further undergoes spontaneous chemical and enzymatic reaction.
Research shows that the number of copper atoms present in laccases
varies in white or yellow laccases and is often substituted by zinc, iron or
manganese atom/molecules (Chen et al., 2015; Haibo et al., 2009; Zhou
et al., 2014).
Laccases are known to catalyze the oxidation of wide range of
phenolic compounds, lignin related compounds and recalcitrant envi­
ronmental pollutants by a radical-catalyzed reaction mechanism (Vis­
wanath et al., 2014). In comparison to bacterial laccases, fungal ones
show higher redox potential oxidizing broad range of substrates. This
difference in redox potential might be due to the structural difference in
T1 copper coordination and chemical nature of the substrate binding
pocket (Piontek et al., 2002). Redox potential are usually found between
430 and 790 mV (Klonowska et al., 2002; Xu et al., 1996). This high
value is due to the coordination of metal ion to the protein backbone,
forcing it to a strained geometry with high redox potential (Riva, 2006).
All laccases are divided into three groups according to their redox po­
tential at T2 site: (1) low E0 enzyme with E0 = 430 mV, (2) middle E0
with potential between 470 and 710 mV, and (3) High E0 enzymes with
redox potential of 730–790 mV (Cambria et al., 2008; Xu, 1997). More
the redox potential, more will be the range of substrates it can catalyze.
Trametes versicolor has the highest redox potential among other laccases,
making it a potential candidate for industrial applications (Zeng et al.,
2017a). Even though fungal laccase show high redox potential, they
have certain limitations at high pH and salt concentrations which can be
overcome with the use of the enzymes from bacterial origin as they can
withstand high salt concentration and can even be activated in its
presence (Singh et al., 2009). Salt inhibition occurs when halide binds to
T1 or T2/T3 which causes inhibition in electron transfer. In the absence
S.F.F. Zofair et al.
Fig. 2. Examples of laccase mediators: (a) Synthetic mediators - 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), hydroxybenzotriazole (HBT),
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), violuric acid (VA), N-hydroxyphthalimide (HPI), N-hydroxyacetanilide (NHA); (b) Natural mediators - vanillin,
acetosyringone, syringaldehyde, p-coumaric acid, ferulic acid, sinapic acid.
of salt, the positive charge on the surface of enzyme hinders the inter­
action between the enzyme and substrate whereas in the presence of
salt, the halide ions tend to bind to the positive charge on the enzyme
which enhances the enzyme-substrate interaction (Li et al., 2019). Ma­
rine bacterial laccases contains highly positive charged residues which
might be due to their habitat and the association of sodium or chloride
ions with some specific loop regions, which is responsible for high cat­
alytic activity in the presence of high salt concentration (Benrezkallah
et al., 2015; Li et al., 2019).
Similarly, laccases can be very strongly inhibited by reagents like
small anions such as azide, halides, cyanide, thiocyanide, sodium azide,
EDTA, thioglycolic acid, detergents, fluoride and hydroxide (Bollag and
Leonowicz, 1984; Jaiswal et al., 2016; Johannes and Majcherczyk,
2000). These reagents inhibit the activity of the enzyme by binding to
the T2 and T3 copper and interrupting the internal electron transfer
process. Other inhibitors which can result in amino acid residue modi­
fications, conformational changes, copper chelation or denaturing of the
laccases include metal ions, fatty acids, sulfhydryl reagents, hydrox­
yglycine, kojic acid, desferal and cationic quaternary ammonium de­
tergents (Gianfreda et al., 1999). Despite the fact that laccases become
inactive in the presence of inhibitors and under various denaturing
conditions, a recent study discovered that pre-incubating laccase with
various organic solvents enhances its activity and thermo-stability sub­
stantially. This increase in activity was due to remodelling at the
3
Journal of Environmental Management 309 (2022) 114676
substrate-binding pocket exerting a more rigid conformation (Radhak­
rishnan et al., 2022; Wu et al., 2019). Furthermore, laccase activity also
increased significantly at 10, 40, and 50 Hz when exposed to a rotating
magnetic field (RMF). This could be due to the presence of paramagnetic
T1 and T2 atom in laccase. The RMF causes change in the copper atom
site which further causes change in its catalytic property (Wasak et al.,
2019). Improved catalytic behaviour of laccase is reported in the pres­
ence of some metal ions and ionic liquids (Ferreira et al., 2021; Zhang
et al., 2015). Metallic ions including Cu+2, Mg+2, K+, Cd+2, Zn+2, Ni+2,
Fe+2 and Mn+2 enhance the catalytic activity of laccase by maintaining
the conformation of its active site (Si et al., 2021). Application of these
methods could expand the use of laccases in various biotechnological
sectors. Besides the industrial application of laccases, they also have the
potential to be used as an anti-proliferative agent towards cancer cells
lines of liver, breast, lungs, stomach and cervix (Chauhan et al., 2019;
Min et al., 2018; Sondhi et al., 2021).
Laccases have been reviewed several times in the past, however not
much discussion has been done with a broad aspect to different immo­
bilization supports and effective mediator compounds for the purpose of
bioremediation. This review aims to highlight the critical parameters
involved in enhancing the catalytic efficiency of laccases and to compile
the newer achievements in this field. In addition, the challenges asso­
ciated with the existing methods along with future directions of research
have also been discussed. We hope this review will provide significant
S.F.F. Zofair et al.
information to the researchers in order to make this enzyme competent
for industrial use.
2. Catalysis by laccase
The presence of three different types of copper atoms in the active
site of laccases, as well as their wide substrate specificity makes these
enzymes highly interesting in studying the mechanism of enzymatic
catalysis. Laccase-mediated catalysis occurs with reduction of molecular
oxygen to water along with the oxidation of the substrate to produce its
corresponding radical. As it produces water as the only by-product, it is
also called as a green catalyst (Riva, 2006; Solomon et al., 1996). Lac­
case activity depends on the pH of the solution, temperature, type of
substrate being oxidized, the presence of mediators and various in­
hibitors like heavy metals. The optimum pH and temperature of laccases
depends on the source of the enzyme. The optimum temperature of most
laccases lies between 30 and 60 ◦ C. The pH influences the stability and
the electrostatic properties of the surface of the protein. Fungal laccases
are generally more stable at acidic pH in the range 2–6, and bacterial
laccase is more stable at pH 5–9 (Guan et al., 2018; Li et al., 2010). Plant
laccases which have hydrogen donor as the substrate are stable at
neutral or alkaline pH (Dwivedi et al., 2011). With an increase in pH, the
hydroxide ion binds to the T2/T3 site, inhibiting the transfer of electrons
from T1 to T2/T3 site and thereby reduces the rate of oxidation of the
substrate (Munoz et al., 1997). These differences in pH and temperature
conditions might account for the diverse functions of the enzyme
(Benfield et al., 1964).
Laccases catalyze the oxidation of their substrates along with
reduction of oxygen to water in three steps:
(i) Reduction of T1 copper atom by accepting electrons from the
substrate and creating its free radical.
(ii) Transfer of electrons from T1 to T2/T3 “tri-nuclear” cluster.
(iii) Reduction of molecular oxygen to water at T2/T3 “tri-nuclear”
structure (Shraddha et al., 2011).
Substrates widely used with laccases include 2,2′ -azino-bis(3-ethyl­
benzothiazoline-6-sulphonic acid (ABTS), 2, 6-dimethoxyphenol (2, 6-
DMP), syringaldazine, guaiacol, catechol, ferulic acid and gallic acid
(Viswanath et al., 2014). Among these substrates, ABTS has been re­
ported to show maximum laccase activity and is thus most commonly
utilized substrate (Li et al., 2008). Among the phenolic substrates, the
enzyme has different preferences for ortho, meta or para substituted
phenols. Ortho substituted compounds (o-phenylenediamine) are
preferred over para (p-phenylenediamine); followed by meta (m-phe­
nylenediamine) substituted compounds (Kumari and SIRSI, 1972;
Thakker et al., 1992). The wide substrate specificity of laccase can be
enhanced by using redox mediators. Redox mediators are low molecular
weight compounds which can be easily oxidized by laccase and produce
unstable reactive radicals which can further oxidize more complex
substrate (Baldrian, 2006; Couto and Herrera, 2007). Mediators act as
electron shuffle and activators of enzyme (Cambria et al., 2008; Li et al.,
2010). Laccases normally have limited oxidation potential for phenolic
compounds. Therefore, some substrates which cannot get oxidized
because of their large size or high redox potential can also be catalyzed
by laccase in the presence of such redox mediators. This makes laccase
an attractive enzyme for industrial applications. Laccases can utilize
natural mediators like acetosyringone, aniline, phenol and synthetic
mediators like ABTS and 1-hydroxybenzotriazole (HBT) for catalysis of
substrates or to enhance the rate of oxidation of the substrate (Christo­
pher et al., 2014) (Fig. 2).
Mediator type and concentration are critical parameters for
employing laccases at the industrial scale. Mediators are costly and in­
crease industrial effluent toxicity. Therefore, determining economically
optimal concentration of mediators is important for its use. Improved
performance was recorded when mediators were used in the
4
Journal of Environmental Management 309 (2022) 114676
degradation of phenolic compounds. Tavares et al. (2012) concluded
that ABTS promoted the highest phenol degradation rate (91%) in
comparison to other mediators or mediator-free laccase system (degra­
dation >80%) (Tavares et al., 2012). Laccase-mediator system (LMS)
involves three different mechanisms for oxidation (Cantarella et al.,
2003):
(i) Electron transfer mechanism; requires a substrate with low
oxidation potential e.g. ABTS (Christopher et al., 2014; Zeng
et al., 2017a, 2017b).
(ii) Radical hydrogen atom transfer (HAT) route, requires a substrate
with weak C–H bond e.g. HBT, violuric acid (VA) (Bourbonnais
et al., 1997; Fabbrini et al., 2002).
(iii) Ionic oxidation e.g. 2,2′ ,6,6’ - tetramethylpiperidine-N-oxyl
(TEMPO) (Bourbonnais et al., 1997; Galli and Gentili, 2004).
Here the rate of oxidation is controlled by thermodynamic driving
force ΔE = E◦ (T1 copper of laccase) − E◦ (N–OH), so laccase with high
E◦ or an E◦ (N–OH) compounds with low E◦ will show higher oxidation
rates or faster electron transfer (Cambria et al., 2008; Xu et al., 1996).
Compounds with high redox potential can only be transformed only if
their redox potential is lowered by chelation, use of mediators or when
the reaction product is subject to an immediately following reaction
(Baldrian, 2006). This occurs when redox mediators can carry out sub­
sequent laccase-catalyzed oxidation and yield more radically active
compounds as observed in the case of ABTS where cation radical ABTS+
gets further oxidized to di-cation ABTS+2 i.e., more than one oxidation
state is observed. Such mediators can oxidize compounds with redox
potential higher than that of the enzymes to be oxidized (Christopher
et al., 2014).
LMS has certain drawbacks as the synthetic mediators commonly
being used are usually toxic, expensive and can inactivate laccase at
concentration above 1 mM, while natural mediators do not show much
higher efficiency (Desai and Nityanand, 2011; Sun et al., 2016). Also,
the presence of mediators in LMS might lead to secondary pollution. To
overcome this limitation, a one-pot process was developed by Sun et al.
(2016) where immobilization of laccase and the mediator Acetylacetone
(AA) is done on chitosan grafted polyacrylamide hydrogel through
laccase-AA initiated polymerization. This process enhanced the half-life
of the enzyme by 3.5 times and maintained its activity even when stored
for 10 months at − 22 ◦ C. Such procedures could cut down the cost by
reusing the mediators along with the enzyme and appears to be highly
promising for industrial purpose.
Another alternative to avoid secondary waste generation could be to
obtain the source of natural mediators and research for complete
oxidation of substrates. This could also prove to open possible cost-
effective approach for use in biotechnology. Apart from the use of me­
diators, studies on site-directed mutagenesis have also been reported to
improve the catalytic efficiency of laccases. Atefeh Khodakarami et al.
(2018) studied T415I and T418I laccase mutants in vicinity to T1 site, as
catalysis of substrate takes place at T1. Increase in catalytic efficiency,
specificity and enhanced thermal stability was obtained in comparison
to the wild-type enzyme (Khodakarami et al., 2018).
Despite these studies on improvements on laccase catalytic effi­
ciency, major limitations in utilization of the enzyme in industries is due
to its inactivation, irreversible denaturation or depletion of the copper
ion from its active site. These limitations can be overcome by immobi­
lization, which offers stability to the enzyme in extreme conditions as
well.
3. Immobilization of laccase and its importance
Even though laccases are highly efficient in treatment of phenolic
contaminants, they do not have high operational stability. Therefore,
immobilization of laccases on supports has become a common technique
for protecting catalytic activity in aqueous solutions and maximizing
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 1
Immobilization of laccase on different supports and its effect on various parameters.
Origin Immobilization surface Immobilization Substrate Optimal Optimal Kinetic parameters, Reference
type pH temperature activity recovery after
immobilization (R),
activity retained on
storage and on
incubation at high
temperature and
reusability

Trametes versicolor Fe3O4 nanoparticles Dopamine self- ABTS 8.0 – Km = 0.161 mM, Vmax = Zhang et al. (2017)
polymerization 0.989 mM min− 1. R =
88.17%. Activity 89%
ͦ
on storage at 4 C after
ͦ
40 days. ~60% at 50 C
after 6 h. 70% after 10
cycles
Trametes pubescens Chitosan bead Entrapment ABTS 4.5 60 >50% activity after 30 Zheng et al. (2016)
days. >50% activity at
ͦ
60–70 C after 2 h >60%
activity after 6 cycles
Aspergillus oryzae Graphene nanosheets Physical Pyrogallol 5.0 50 17% activity after 4 Skoronski et al.
adsorption cycles (2017)
Covalent binding 6.0 30 80% activity after 6
cycles
Pycnoporussanguineus Immobead-150 Covalent binding m-cresol 3.0 70 R = 97.1%. >90% Gonzalez-Coronel
ͦ
CS43 activity at 50 C after 2 h. et al. (2017)
89% activity after 5
cycles of reuse
Pycnoporussanguineus LentiKat Entrapment m-cresol 4.0 50 Km = 4.71 mM, Vmax = Gonzalez-Coronel
CS43 0.4 mM h− 1. R = 89% et al. (2017)
ͦ
91% activity at 50 C
after 24 h >90% activity
after 5 cycles
Streptomyces Copper alginate beads Entrapment – – – 50% activity after 8 Niladevi and Prema
psammoticus cycles (2008)
Trametes versicolor Polyamide 6/chitosan BSA as spacer + ABTS – – R = 6.4 ± 1.3%. 55% Maryšková et al.,
ͦ
nanofibers glutaraldehyde activity at 4 C after 14 2016
cross-linking days. <50% activity
after 3 cycles
HMD as spacer + R = 7.3 ± 1%. 81%
ͦ
glutaraldehyde activity at 4 C after 14
cross-linking days. <50% activity
after 3 cycles of reuse
White-rot fungi Micro-mesoporous Adsorption ABTS 4.0 40 Km = 0.217 ± 0.005, Pang et al. (2016)
Zirconium-metal Vmax = 0.072 ± 0.009
organic framework (Zr- mM min− 1. R = 95.90 ±
MOF) 0.28%. 55.4% activity at
ͦ
4 C after 21 days. ~40%
ͦ
activity at 50 C after 1 h.
65.5% activity after 5
successive cycles of use
ͦ
at 40 C
Trametes versicolor Halloysite nanotubes Covalent binding ABTS 5.0 60 Km = 90 μM, Vmax = 41 Kadam et al. (2017)
functionalized with μM min− 1. R = 90.20%
ͦ
aminosilane 87% activity at 4 C after
30 days. 91% activity at
ͦ
60 C after 6 h. 67%
activity after 12
successive cycles of use
Myceliophtho-ra Epoxy-functionalized Covalent binding ABTS 3.0 – Km = 25.3 μM, Vmax = Mohammadi et al.
thermophile silica particles 1.56 μM min− 1 96% (2018)
ͦ
activity at 35 C after 1 h.
61% activity after 5
cycles of use
Trametes versicolor Amine functionalized Adsorption ABTS 6.0 – Km = 116.5 μM L− 1, Lin et al. (2017b)
magnetic Fe3O4@C Vmax = 343.6 μM/(mg.
ͦ
nanoparticles min). 70% activity at 4
C after 30 days. >60%
activity after 10 cycles of
use
Trametes versicolor Cu (II)-chelated Adsorption Syringalda-zine 5.5–6.0 30–40 Km = 0.070 mM, Vmax = Alver and MetinÜ,
chitosan nanoparticles 0.14U mg− 1. R = 52.6%. 2017; Alver et al.,
50 ± 0.62% activity 2017
after 8 successive cycles
of use
White-rot fungi Adsorption ABTS 4.0 50 Zhong et al. (2017)
(continued on next page)

5
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 1 (continued )
Origin Immobilization surface Immobilization Substrate Optimal Optimal Kinetic parameters, Reference
type pH temperature activity recovery after
immobilization (R),
activity retained on
storage and on
incubation at high
temperature and
reusability

Cu–MOF (metal Km = 0.157 mM L− 1,

organic framework) Vmax = 0.058 mM
min− 1. R = 95.2%.
ͦ
18.8% activity at 4 C
after 3 weeks. 48.4%
activity after 7 cycles of
use
Aspergillus Green coconut fiber Covalent binding ABTS – – Km = 0.0717 mM, Vmax Cristóvão et al.,
= 0.247 mM min− 1 2012
(without reduction). R
= 50%. 10% activity at 4
ͦ C after 24 h ~50%

activity after 3 cycles of

use (pH = 10)
Myceliophthora Bacterial nanocellulose Physical ABTS catechol 3.0 50 Km = 0.77 mM, Vmax = Sampaio et al.
thermophila membrane from adsorption DMP 7.0 50 5.29 mM min− 1 (ABTS (2016)
Gluconacetobac-ter 6.0 60 as substrate). R = 70%.
ͦ
xylinum 80% activity at 50 C
after 1 h
P. ostreatus (crude) Functionalized TiO2 Covalent binding ABTS 3.0 50 Km = 42.9 ± 3.3 μM, Ji et al. (2017)
nanoparticle (GA coupling) Kcat = 75.5 ± 9.4 μmol
min− 1 mg− 1. R = 125.8
± 15.2%. 75% activity
ͦ
at 25 C after 30 days.
ͦ
85% activity at 50 C
after 2 h. 25% activity
after 5 cycles of use
ͦ
Pleurotus ostreatus Eupergit® Covalent binding Syringalda-zine 5.8 50 98% activity at 25 C Hublik and
C after 10 days. Maximum Schinner (2000)
stability at pH 10
Carica papaya leaves Chitosan Entrapment ABTS 8.0 80 Km = 0.14 mM, Vmax = Jaiswal et al.
0.02 μmol min− 1 ml− 1. (2016)
R = 98%. 80% activity at
ͦ
4 C after 30 days.
ͦ
~300% activity at 80 C
after 90 min–40%
activity after 3 cycles of
use
Trametes sp. Bacterial cellulose from Adsorption ABTS 4.0 70 Km = 0.94 mM, Vmax = Drozd et al. (2018)
Komagataeibact-er 7.24 μmol min− 1 l− 1.
xylinus exposed to 39.1 ± 2.1% activity at
ͦ
rotating magnetic field 60 C after 30 min. 70%
activity after 7
successive cycles of use
Trametes versicolor Nanoporous gold Covalent coupling, 2,6- – – Km = 0.17 mM, Vmax = Qiu et al. (2009)
(NPG) electrostatic dimethoxyphenol 1.19 mM min− 1 (for
attraction, (DMP) NPG at micron scale).
physical Physical adsorption
adsorption showed best results
ͦ
Trametes pubescens Chitosan bead Entrapment ABTS 5.0 60 40% activity at 4 C after Zheng et al. (2016)
30 days. >80% activity
ͦ
at 25 C after 72 h >50%
ͦ
activity at 60–70 C after
2 h. 60% activity after 6
cycles of successive use
Trametes versicolor Methacrylyol Adsorption ABTS 5.0 60 Km = 113.3 μM L− 1, Lin et al. (2017a)
functionalized super Vmax = 338.9 μmol mg− 1
ͦ
paramagnetic min− 1. 60% activity at 4
Fe3O4@SiO2 C after 30 days. 62%
ͦ
activity at 60 C after
150 min > 80% activity
on reusing it to
decolorize methyl red
for 7 days
Pycnoporussanguineus Titania nanoparticles APTES and GA ABTS 3.0 50 Km = 21.24 ± 1.92 μM. García-Morales
CS43 coupling R = ~100%. 90% et al. (2018)
ͦ
activity at 40 C after 72
h
Trametes versicolor Covalent binding ABTS 4.0 40 Das et al. (2017)
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Table 1 (continued )
Origin Immobilization surface Immobilization Substrate Optimal Optimal Kinetic parameters, Reference
type pH temperature activity recovery after
immobilization (R),
activity retained on
storage and on
incubation at high
temperature and
reusability

Magnetic iron Km = 0.58 mM, Vmax =

nanoparticles 0.25 mM min− 1 87%
activity after 5 cycles of
use
T. versicolor Nano-porous silica Adsorption 2,4-DNP 5.0 40 Km = 0.013, Vmax = 0.38 Dehghanifard et al.
beads μmol min− 1 >85% (2013)
activity after reusing for
30 successive days
Trametes versicolor Magnetic nanoparticles Covalent bonding Catechol 4.0 45 Km = 70.15 ± 5.33 μM, Sánchez-Ramírez
coated with chitosan Vmax = 2.00 ± 0.15 et al. (2018)
(C-MNP) μmol min− 1 50%
ͦ
activity at 4 C after 28
days. >55% activity at
ͦ
50 C after 4 h. 50%
activity after 5 cycles of
use.
Trametes versicolor Cu2+− chelated Metal affinity ABTS 6.0 25 Vmax = 0.095 mmol L− 1 Wang et al. (2012)
magnetic mesoporous adsorption min− 1. 72.6%
silica nanoparticles degradation of phenol
was obtained after 10
cycles of reuse
Lentinula edodes Chitosan Adsorption DMP 4.0 60 Km = 256 μM, Vmax = D’Annibale et al.
85.5 μmol min− 1 mg− 1. (1999)
R = 45%. 60% activity
ͦ
at 5 C after 6 months.
80% activity after 30
cycles of reuse
DeniLite II S from Polydopamine- Physical ABTS 4.0 30 Km = 0.164 mM, Vmax = Cao et al. (2018)
genetically modified modified 3D adsorption 2.924 mM min− 1. R =
Aspergillus interconnected 40.8%. 94.1% activity at
ͦ
macroporous silica 4 C after 15 days. 83%
ͦ
activity at 40 C after 2.5
h. 85.5% activity after 5
cycles of reuse
Ascomycete Polyvinylidene fluoride Cross-linking ABTS – – Km = 62.2 ± 12.5 μM, Jahangiri et al.
Phoma sp. strain UHH (PVDF) membranes through electron Vmax = 1.4 ± 0.6 Umg− 1. (2018)
ͦ
5–1-03 beam irradiation 43% activity at 4 C after
36 days. 62% activity
after 10 cycles of reuse
Coprinus comatus Maple biochar Adsorption ABTS – – Km = 2.68 mM. R = Li et al. (2018)
(mushroom) 66.5%. ~30.3% activity
ͦ
at 60 C after 6 h. 33.8%
activity after 7 cycles of
use
CotA gene from Cu(II) chelated Adsorption ABTS 5.0 80 Km = 1.62 mM, Vmax = Samak et al. (2018)
B. subtilis magnetic graphene 9.7 μM min− 1 mg− 1.
ͦ
oxide nano-sheet 80.7% activity at 4 C
after 32 days. 80%
ͦ
activity at 80 C after 3 h.
89.4% activity after 10
cycles of use
Trametes versicolor TiO2 nanoparticles Physical ABTS – – Km = 92.7 ± 10.7 μM. R Hou et al. (2014)
adsorption = 48 ± 6%. ~50%
ͦ
activity at 25 C after 20
days
Trametes versicolor TiO2 nanoparticles sequential ABTS – – Km = 56.0 ± 3.0 μM. R Hou et al. (2014)
immobilization = 79 ± 6%. ~90%
ͦ
activity at 25 C after 20
days
ͦ
Aspergillus oryzae Plasma-coated Cross-linking ABTS 6.0 – ~18% activity at 4 C Lee et al. (2018)
nonwoven followed by needle after 15 days. 40%
ͦ
polyethylene/pol- punching and activity at 50 C for after
ypropylene (PEPP) thermal bonding 120 min. 20% activity
fibers (plasma (NPTB) after 8 cycles of use
polymerization by
cyclopropylami-ne)
T. pubescens Cui 7571 Chitosan Cross-linking with ABTS 4.0 55 Km = 127.6 μM 57.14% Ma et al. (2018)
ͦ
genipin activity at 4 C after 30
days. 63.82% activity at
(continued on next page)

7
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 1 (continued )
Origin Immobilization surface Immobilization Substrate Optimal Optimal Kinetic parameters, Reference
type pH temperature activity recovery after
immobilization (R),
activity retained on
storage and on
incubation at high
temperature and
reusability
ͦ
65 C after 12 h.
>55% activity after 11
cycles of use
Trametes versicolor Poly(hydroxyethyl Physical Syringalda-zine 6.0 45 R = 73%. 72% activity Bayramoglu et al.
ͦ
methacrylate-co- adsorption at 4 C after four weeks. (2019)
ͦ
vinylene carbonate), p 66% activity at 55 C
(HEMA-co-VC) after 120 min.
microbeads 51% activity after 10
successive cycles
Trametes versicolor Sepharose linked Covalent binding ABTS 3.0 65 Km = 55 μM, Vmax = Zofair et al. (2020)
antibody followed by 4.098 mM min− 1. R =
ͦ
bioaffinity 90%. 75% activity at 4 C
after 30 days.
ͦ
60% activity at 50 C
after 2 h. 44% activity
after 10 cycles of use
ͦ
Trametes versicolor Core/shell Fe3O4/ Covalent binding ABTS 4.0 60 >50% activity at 4 C Chen et al. (2021a,
nylon composite after 21 days. Approx. 2021b)
nanoparticles 35% activity after 10
cycles of use
Pleurotus ostreatus Monoaminoethyl-N- Ionic adsorption ABTS 5.0 55 Km = 0.15 ± 0.02 mM, Brugnari et al.
aminoethyl Vmax = 3.85 ± 0.29 mg (2018)
(MANAE–agarose) L− 1 min− 1. R = 138%.
ͦ
80% activity at 4 C after
40 days. 50% activity at
ͦ
55 C after 180 min. 90%
activity after 10
successive cycles of use
ͦ
Trametes versicolor Pinewood nanobiochar Covalent binding ABTS 3.0 – 50% activity at 4 C after Naghdi et al.,
30 days. 70% activity at 2018a, 2018b
ͦ
50 C after 8 h. 30%
activity after 3
successive cycles of use
ͦ
Trametes maxima Amino- functionalized Covalent binding ABTS 4.0 60 94.32% activity at 4 C Suman et al. (2018)
chicken feather (GA crosslinking) after 3 weeks
ͦ
particles (ACFP) 75% activity at 50 C
after 2 h. 95% activity
after 8 cycles of use
ͦ
Trametes versicolor Magnetic biochar Adsorption ABTS 3.5 45 81.8% activity at 4 C Zhang et al.
nanoparticles followed by GA after 30 days. 64.4% (2020a, 2020b)
ͦ
crosslinking activity at 25 C after 30
days. 85% activity after
seven cycles of use.
ͦ
Trametes versicolor Polyamide 6 nanofibers Adsorption ABTS 7.0 – 50% activity at 4 C after Maryskova et al.
followed by GA 30 days. 88% activity (2019)
crosslinking after 5 cycles of reuse
Trametes versicolor poly(methyl Adsorption ABTS 5.0 25 Km = 0.098 ± 0.009 Jankowska et al.
methacrylate)/ mM, Vmax = 0.029 ± (2020)
polyaniline fibres 0.002 U mg− 1. 86%
activity when stored for
30 days.
66% activity after 10
cycles of use
Covalent binding Km = 0.119 ± 0.020
mM, Vmax = 0.021 ±
0.002 U mg− 1. 95%
activity when stored for
30 days.
73% activity after 10
cycles of use
Trametes pubescens Ca-alginate beads Entrapment with ABTS 4.0 40 Km = 0.325 mM, Vmax = (Lassouane et al.,
crosslinking 1.296 mM min− 1 96.2% 2019)
ͦ
activity at 4 C after 35
days. >90% activity at
ͦ
40 C after 24 h >70%
removal of BPA after 10
successive cycles
Trametes versicolor Amino-modified Two step ABTS 4.0 40 Km = 4.73 × 10− 5mol/L, Yang et al. (2021)
mesoporous silica Crosslinking R = 59.6%. 56% activity
(continued on next page)

8
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 1 (continued )
Origin Immobilization surface Immobilization Substrate Optimal Optimal Kinetic parameters, Reference
type pH temperature activity recovery after
immobilization (R),
activity retained on
storage and on
incubation at high
temperature and
reusability

after 5 consecutive
cycles
White rot fungi Bacterial Nanocellulose Physical ABTS – – Km = 0.53 ± 0.06 mM, Yuan et al. (2018)
(K. Xylinus DHU-ZCY- adsorption Vmax = 2.74 ± 0.13 mM
1) min− 1 ~70% activity at
ͦ
70 C after 1 h.
91.1% activity after 10
consecutive cycles
Aspergillus oryzae Magnetic nanoparticles Covalent binding ABTS – – Km = 0.0062 mM, Vmax Fortes et al. (2017)
(Genetically coated with silica = 0.062 mM min − 1
ͦ
modified) 30% activity at 4 C after
ͦ
35 days. ~ 40% at 60 C
after 6 h. 75.8% activity
after 6 cycles of use
Trametes versicolor Mineral hybrid Entrapment ABTS 4.6 40 Km = 273.32 μM, Vmax = (Zhang et al., 2021)
nanoflowers 96.43 μM min− 1 93.2%
activity when stored at 4
ͦ C for 30 days.

Mineral hybrid Km = 337.15 μM, Vmax =

nanopetals 88.73 μM min− 1 83.7%
activity when stored at 4
ͦ C for 30 days.

their stability over broad range of operating conditions. Reports have
suggested that the use of free enzymes in traditional enzyme applica­
tions has many drawbacks like non-reusability, loss of stability and is not
favourable economically which can be overcome by immobilization
processes (Arsalan et al., 2020; Lin et al., 2017a; Ping et al., 2008). Thus,
immobilization of laccase on insoluble supports is an efficient way to
improve its stability and permit its reuse in reaction system. Immobili­
zation of laccases has been done on various supports including nano­
particles, membranes, biological polymers, mesoporous material,
porous glass, microsphere, charcoal, activated carbon and nanotubes.
The activity of immobilized laccase depends largely upon the
immobilization procedure, immobilization time and the properties of
the carrier being used (Lin et al., 2017b). Various techniques like surface
binding, gel entrapment (hydrogel), entrapment in reverse micelles,
covalent and cross-linking bonding between an enzyme and matrix have
been commonly used for improvement in the operational stability of the
enzyme (Fernández-Fernández et al., 2013; Sun et al., 2016; Wang et al.,
2013). However, the use of biological polymers in immobilization has
added benefits like non-toxicity, economical and biocompatibility.
Alginate and chitosan involving microencapsulation technology are
commonly being employed in immobilization due to their biodegrad­
able property (Khan et al., 2020). Increased stability was observed when
laccase was immobilized on chitosan based support, as chitosan provides
external backbone for the enzyme which protects it against conforma­
tional changes and provides resistance to inactivation (Jaiswal et al.,
2016; Lu et al., 2007). A novel method for encapsulation of laccase on
chitosan-nanobiochar matrix was reported, where the biochar supports
adsorption of pollutants and provides enough contact time for biodeg­
radation (Naghdi et al., 2019). Similar green-technology based biocat­
alyst like paper-waste derived α cellulose-fibrils with super-magnetic
and functionalized chitosan used as a support for immobilization of
laccase showed exceptionally high activity of 71% after 11 cycles of use
(Ghodake et al., 2018).
Another relatively simple and inexpensive method of laccase
immobilization is via entrapment. Lassouane et al. (2019) studied
crosslinked-entrapped laccase which reduced leaking from
calcium-alginate beads by 7-fold and also provided resistance to inac­
tivation of laccase and prolonged its shelf-life. Membrane modules have
been also applied to the immobilization of laccase resulting in improved
mechanical strength, reusability and prolonged life. The enzyme
immobilized on polysulfone membrane with functionalized
carbon-nanotubes (CNT) showed easy recovery and was effective in
treatment of phenolic compounds (Costa et al., 2019). This support can
be reused for enzyme re-immobilization and has its potential application
in industries as a biocatalyst. Laccase activity can also be enhanced and
stabilized prior to immobilization by using metal ions chelated to
polymer network. Chelated metal ions are capable of binding through
coordinative interaction with some functional groups exposed on the
enzymatic surface (Baig et al., 2015). When copper (II) chelated
chitosan-graft-poly (glycidyl meth-acrylate) nanoparticles was modified
with polyethylene imine (PEI), it showed increased affinity for laccase
and increased loading capacity. PEI bound to the support acts as a
spacer, thereby reducing the steric hindrance and providing increased
catalytic efficiency (Alver and MetinÜ, 2017).
With the development of nanostructured materials with larger sur­
face area, a series of nanomaterials with different sizes and shapes have
been used as substrates for enzyme immobilization (Arsalan and You­
nus, 2018). Among them, Fe3O4 magnetic nanoparticles have emerged
as promising supports owing to their large surface area, low
mass-transfer resistance and well-defined surface properties. Moreover,
enzymes immobilized on Fe3O4 magnetic nanoparticles are not only
capable of maintaining their own unique performance, but are also easy
to separate and recycle. Wang et al. (2013) studied immobilization of
laccase with Fe3O4/SiO2 nanoparticles with size less than 30 nm
resulting in large surface area and high loading capacity. Immobilization
with this support caused decolourization of azo dyes within an hour in
the presence of mediator, the process which usually takes 6–24 h in the
absence of mediator compound. Adsorption of dye by the Fe3O4/SiO2
nanoparticle support helped in enhancing the degradation of dye (Wang
et al., 2013). Silica-coated magnetic nanoparticles can be regenerated
for enzyme immobilization with good yield. These nanoparticles can
easily be recovered by applying external magnetic field (Moldes-Diz
et al., 2018). Similarly, Kadam et al. (2017) studied the combined
supramagnetic plus aminosilanization property into halloysite nano­
tubes (HNT) which provides excellent separation of supporting material
and effective laccase loading, respectively, in order to catalyze

9
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 2
Bioremediation of different pollutants in the presence of laccase-mediator system (LMS).
Source of laccase Pollutant Mediators optimized Effective mediator Mediator pH Optimum Reference
amount Tempera-
ture (oC)

Trametes versicolor Isoproturon HBT, VA, ABTS, Acetosyringone, HBT, VA 1.0 mM 5.0 50 Zeng et al. (2017a)
Vanillin, Syringaldehyde
Aspergillus oryzae Diclofenac – Syringaldehyde 100 μM 3.0, – Lloret et al. (2012)
(Genetically 4.5
modified)
Trametes versicolor Sulfonamides – Syringaldehyde 1.0 mM – – Becker et al. (2016)
Tetracyclins 10 μM
Trametes versicolor Sulfamethoxazole ABTS, Syringaldehyde, Guaiacol Guaiacol 6.0 – Kadam et al. (2017)
Trametes versicolor Indomethacin, – ABTS, HBT 1.0 mM 4.5 – Tran et al. (2010)
Naproxen
Trametes versicolor BPA – ABTS – 7.0 – Pang et al. (2015)
Trametes trogii Azure B HBT, p-hydroxybenzoic acid, HBT 0.5 mM 4.5 30, Grassi et al. (2011)
Phenol, Methionine, Tyrosine, 50
Trametes trogii Xylidine Anisaldehyde, Vanillin, Vanillic HBT 50 Grassi et al. (2011)
acid, Ferulic acid
Trametes versicolor Malachite green – Acetylacetone 6.0 – Sun et al. (2016)
Rigidoporus lignosus 2-methylanthracene HBT, VA, ABTS VA 0.9 mM 4.5 – Cambria et al. (2008)
Trametes versicolor Phenol red Imidazole, Sulfanilic, 4-dimethy­ Glycine 1.0 mM 4.0 40 (M. Gomaa, 2005)
lamino benzaldehyde, Ampyrone,
Thiamine, Glycine, Glutamic,
Nicotinic acid, Cysteine
Trametes versicolor Acenaphthene, Methionine, Cysteine, Tyrosine, HBT, Tyrosine, 1.0 mM 4.5 – (Johannes and
Acenaphthylene Tyrosine hydrazide, Reduced Reduced Glutathione Majcherczyk, 2000)
Glutathione
Trametes sp. Methyl orange Acetosyringone, VA, HBT, TEMPO VA 0.1 mM 4.2 Wang et al. (2018b)
Methyl blue
Trametes versicolor Carbamazepine – ABTS 18 μM 5.5–6.0 45 Naghdi et al. (2018b)
Trichoderma Malachite green, – HBT 2.0 mM 6.0 – Bagewadi et al. (2017)
harzianum Methylene blue
Coriolopsis gallica BPA – HBT 1.0 mM 5.0 – Daâssi et al. (2016)
Trametes versicolor Salicylic acid, – HBT 1.0 mM 4.5 – Nguyen et al. (2014)
enterolactone and
oxybenzone
Pichia pastoris Indigo dye 3,5-dimethoxy-4- Methyl 3,5-dime­ 120 μM 7.5 60 Yin et al. (2019)
(mutant-PIE5) hydroxybenzaldehyde, Methyl thoxy- 4-
3,5-dimethoxy- 4- hydroxybenzoate
hydroxybenzoate, ABTS, Syringic ABTS 200 μM 7.0
acid, HBT
Trametes versicolor Methylene blue – HBT 5.0 mM 4.5 – (Forootanfar et al.,
2012)
Trametes versicolor Benzo[a]pyrene – ABTS 1.0 mM 4.0 40 Li et al. (2010)
Trametes versicolor Phenol red – HBT 10 mM 5.0 – Moldes et al. (2004)
Grifola frondosa BPA – HBT 2.0 mM 4.0 – (NITHERANONT
et al., 2011)
Pleurotus eryngii 2,4-dichlorophenol ABTS, HBT ABTS, HBT 1.0 mM 5.0 – Rodríguez et al.
Benzo[a]pyrene HBT (2004)
Pleurotus ostreatus Perfluorooctane- – HBT (Cu2+) 20 μM 4.9 – Luo et al. (2018)
sulfonate HBT (Mg2+) 6.5
Trametes versicolor Pesticides ABTS, Caffeic acid, Chlorogenic Vanillin 1.8 mM 5.0 – Kupski et al. (2019)
acid, p-coumaric acid, Ferulic
acid, Gallic acid, Protocatechuic
acid, Vanillin
Streptomyces Acid orange 63 Syringaldehyde, Acetosyringone, Acetosyringone, 0.5 mM 8.0 60 Blánquez et al. (2019)
ipomoeae CECT Methyl syringate Methyl syringate
3341
(recombinant)
Trametes versicolor Pyrimethanil, Guaiacol, HBT, VA, Violuric acid 4.0 mM 4.0 35 (Jin et al., 2016)
Isoproturon Syringaldehyde, Acetosyringone,
Chlorpyrifos ABTS, Vanillin Vanillin 5.0 30
Atrazine HBT 4.0
Chlorothalonil Acetosyringone 5.0
Streptomyces Indigo carmine – β-(10-phenothiazyl)- 50 μM 8.0 – Coria-oriundo et al.
ipomoeae Malachite green propionic acid 250 μM (2021)
(recombinant)
Trametes versicolor Sulfadimethoxine Soyabean meal extract, HBT, p- Soyabean meal – – – Liang et al. (2017)
Coumaric acid, ABTS extract (vanillin,
apocynin, and
daidzein)
Trametes versicolor 3,5-dichloroaniline Catechol, Syringaldedyde, Catechol 2.0 mM 5.0 – Sarker et al. (2020)
Syringic acid, Caffeic acid, Gallic
acid
Trametes versicolor 0.5 mM 4.5 – (Chen et al., 2021b)
(continued on next page)

10
S.F.F. Zofair et al. Journal of Environmental Management 309 (2022) 114676

Table 2 (continued )
Source of laccase Pollutant Mediators optimized Effective mediator Mediator pH Optimum Reference
amount Tempera-
ture (oC)

Guaiacylglycerol- ABTS, Syringaldehyde, Syringaldehyde,

β-guaiacyl ether Acetosyringone, Methyl syringate Acetosyringone,
Methyl syringate
Aspergillus aculeatus Olsalazine ABTS, HBT, p-Coumaric acid ABTS 1.0 mM 5.0 – Olusegun et al. (2021)
Obba rivulosa Veratryl alcohol HBT, TEMPO, ABTS, VA, HPI, TEMPO 3.0 – Kontro et al. (2020)
(OrLcc1 & OrLcc2) β-O-4-dimer Methyl syringate, Syringyl nitrile VA
Funalia trogii, BPA – Butylatedhydroy- – 5.5 50 Atacag Erkurt (2015)
Trametes toluene
versicolor
Trametes sp. Anthracene – ABTS 1.0 mM – – Wu et al. (2008)
Bacillus subtilis Aflatoxin B1 Methyl syringate, Caffeic acid, Methyl syringate 1.0 mM 6.0–10.0 40–80 Wang et al. (2019)
(CotA) Zearalenone Syringaldehyde, HBT, Vanillin, 5.0–10.0 20–80
Gallic acid, p-Coumaric acid
Trametes versicolor Octylphenol ABTS, HBT, TEMPO TEMPO 0.16 mM 5.0 50 (Wang et al., 2021b)
polyethoxylate
Trametes versicolor Polyurethanes – HBT 0.2 mM 4.5 – Magnin et al. (2021)
Streptomyces Ciprofloxacin, Methyl syringate, Syringaldehyde, Acetosyringone 0.5 mM 8.0 – Blánquez et al. (2016)
ipomoeae (SilA) Norfloxacin Acetosyringone, p-
Hydroxybenzoic acid
Pleurotus Aflatoxin B1 ABTS, TEMPO, Acetosyringnone, Acetosyringnone 0.5 mM 7.0–8.0 60 (Song et al., 2021)
pulmonarius Zearalenone Syringaldehyde ABTS 4.0–8.0 50
(Lac2)
Trametes versicolor Remazol blue RR, Acetosyringnone, Syringaldehyde, Syringaldehyde 50 μM 3.0–5.0 – (Mendoza et al., 2011)
Red FN-2BL, ABTS, p-Coumaric acid, VA, N-
Blue, Red BWS Hydroxyphthalimide, 3-Hydrox­
yanthranilic acid, Vanillin
Trametes versicolor Rem blue RR 2, 6-DMP, Phenol, Vanillin, Vanillin 0.5 mM 5.5 40–70 Şaşmaz et al. (2011)
Guaiacol, Veratryl alcohol, L-
Tyrosine
Trametes trogii Textile effluent 2, 6-DMP, HBT, Syringaldehyde, HBT 1.0 mM 5.0 50 (Khlifi et al., 2010)
Syringate, Vanillin, Vanillate,
Acetosyringone, m-, o- and p-
Coumarate
Paraconiothyrium Alprazolam and ABTS, 2,6 DMP, HBT, Vanillic acid HBT 2.0 mM 5.0 – Ostadhadi-Dehkordi
variabile Diazepam et al. (2012)
Trametes versicolor Chlorpyrifos ABTS, HBT, VA, 2, 6-DMP, Vanillin 2.0 mM 5.0 – Xie et al. (2013)
(recombinant) Vanillin, Guaiacol
Trametes versicolor Oxybenzone ABTS, 4-Hydroybenzoic acid, ABTS (synthetic) 1.0 mM 6.0 – Garcia et al. (2011)
HBT, Acetosyringone, Sinapinic Acetosyringone
acid, p-Coumaric acid, Ferulic (natural)
acid, Syringaldehyde
Trametes versicolor N,N-diethyl-m- – ABTS 1 μM 4.5 – Tran et al. (2013)
toluamide HBT 0.01 mM
Ganoderma lucidum Triclosan ABTS, HBT, Syringaldehyde, HBT, Syringaldehyde 1.0 mM 4.0 – Murugesan et al.
Acetovanillone, Vanillin, p- (2010)
Coumaric acid, 2,4- DMP,
Guaiacol
Perenniporia strain Sulfonamide 4-Hydroxybenzyl alcohol, 4- ABTS, VA 1.0 mM 4.1 – Weng et al. (2012)
TFRI 707 hydroxyacetophenone,
Syringaldehyde, 4-Cyanophenol,
ABTS, VA
Brassica juncea L. Methyl orange ABTS, HBT, Acetosyringone, ABTS 10 mM 8.0 – Telke et al. (2010)
Vanillin, Syringaldehyde, DMP,
Syringic acid, Hydroquinone,
Pyrogallol

biodegradation of micropollutant sulfamethoxazole. Laccase was effi­
ciently loaded on Aminopropyltriethoxysilane-magnetic-Halloysite
nanotubes-Glutaraldehyde (A-M-HNTs-GTA) which enhanced thermal
and storage abilities and offered high enzyme loading capacity, making
it a good choice for practical applications (Kadam et al., 2017).
Immobilization of enzymes causes a frequent decrease in activity
because the enzyme moves from a free state to a fixed state that reduces
the liberty of movement and sometimes the space of the enzyme
(Arsalan and Younus, 2018). This explains an increase in Km value
observed after immobilization, indicating lower affinity for substrate
attributable to diffusional limitations (Lu et al., 2007). Several problems
are related to bioavailability of laccase in continuous operations due to
diffusional limitations and difficulty in enzyme regeneration. Surface
immobilized enzymes can be used as a whole-cell catalyst by using
compatible bacterial host to minimize the mass transfer limitations.
Wang et al. (2012) developed a regenerative engineered bacterial sys­
tem with laccase immobilized on the surface of P. putida cells. The re­
sults showed regenerability and elimination of mass transfer limitations.
The diffusional limitations could also be minimized with the use of
spacers. This was observed when Sepharose-linked anti-laccase antibody
support was employed for immobilization, which exhibited an effec­
tiveness factor of 0.9 with high thermal stability and minimized diffu­
sional limitations (Zofair et al., 2020). Such support with the use of
spacers can minimize the steric hindrance often encountered with high
molecular weight substrates. The increased resistance to thermal dena­
turation after immobilization of laccase offers a potential advantage in
wastewater treatment applications. Liquid supports based on an aqueous
biphasic system have also demonstrated great catalytic efficiency and
reusability in addition to solid supports (Ferreira et al., 2021). Laccase
immobilization on various surfaces and the effect of immobilization on

11
S.F.F. Zofair et al.
different parameters is shown in Table 1.
Besides these immobilization systems, few studies on co-
immobilization of laccase and its mediators have been reported which
allowed for the simultaneous recovery of enzyme and mediator thereby
increasing the efficiency and minimizing secondary waste generation
(Xue et al., 2020; Yaohua et al., 2019). Such strategies will make the
bioremediation process more feasible.
4. Role of laccase in bioremediation
Bioremediation process involves the use of metabolic capacity of
distinct species of microorganisms to clean up the environment (Wata­
nabe, 2001). The process depends upon the environmental factors and
the nature of chemicals in order to degrade toxic contaminants from the
environment (Boopathy, 2000). As already mentioned, degradation by
physical and chemical method are either time consuming, expensive or
produce secondary pollution and thus are not much efficient for treat­
ment of highly contaminated industrial effluents (Zeng et al., 2017b).
Therefore, use of enzymes has increasing attention in bioremediation of
recalcitrant environmental pollutants because of their high efficiency,
selectivity and environment-friendly reactions. Among these, the major
class of enzymes used for bioremediation belong to extracellular fungal
peroxidases such as lignin peroxidase, manganese peroxidase and fungal
laccases. The use of these enzymes is restricted as they require the
external supply of hydrogen peroxide as cofactors for the oxidation of
variety of substrates (Huber et al., 2018). However, among these, lac­
case requires only molecular oxygen to carry out oxidation of its
Fig. 3. Laccase in treatment of toxic environmental pollutants.
12
Journal of Environmental Management 309 (2022) 114676
substrate. Laccase have enormous potential as biocatalysts in pulp and
paper bleaching, waste water treatment, soil bioremediation, on-site
waste destruction and it can also be used as biosensors to detect the
presence of toxic substance.
Table 2 shows bioremediation of several chemicals and dyes in in­
dustrial effluents using laccase. The influence of several mediator sys­
tems is summarized along with their working concentration, pH and
temperature.
4.1. Bioremediation of pollutants in soil
Polyaromatic hydrocarbons (PAHs) and xenobiotics are the major
contaminants present in soil which can be removed by laccase. Sixteen
PAHs are included among U.S. Environmental Protection Agency (EPA)
priority pollutants− (Goodale, 2015). They are mutagenic and carcino­
genic compounds. High molecular weight PAH are less soluble and it
cannot be degraded by bacteria present in soil and thus its environ­
mental persistence increases (Wilson and Jones, 1993). PAH contami­
nated soil can be treated with fungi secreting laccase. Li et al. (2010)
reported the use of laccase in bioremediation of aged polluted soil
contaminated by gas plant for degradation of PAHs-benzopyrene,
anthracene and benzoanthracene. Here the addition of mediators
enhanced the rate of degradation but not as efficient as observed in
liquid cultures. This might be because laccase-mediators do not work
efficiently in environment lacking water. Hence a packed bed reactor
with continuous flow of laccase and mediator can be a promising
approach in treating pollutants in water lacking environment (Vipotnik
S.F.F. Zofair et al.
et al., 2021). Also some methods are thought to be impotent for biore­
mediation because laccase does not work well in alkaline condition
which is present in most of the soil (Li et al., 2010). Cambria et al. (2008)
reported Rigidoporus lignosus produces some aromatic compounds which
could act as natural mediators for oxidation of PAHs anthracene and
2-methyl anthracene. Besides PAHs; BPA, EDCs and contaminants pre­
sent in bottom sediments can also be treated efficiently with laccase.
BPA is a major component present in various consumer products like
plastic and detergents. Commercial enzymes are very expensive to use at
an industrial scale for treatment of BPA. Atacag Erkurt (2015) studied
comparative degradation of active crude extract (ACE) from Funalia
trogii, commercial laccase from T. versicolor and the mixture of inacti­
vated crude extract with pure commercial laccase. The study concluded
that the mixture used could completely degrade BPA in 4 h incubation as
compared to ACE and commercial laccase. The presence of activator or
mediator molecules like butylhydroxytoluene present in the crude
extract offers protective effect to laccase and helps to enhance the lac­
case catalyzed biotransformation of BPA. Zeng et al. (2017a, 2017b)
reported simultaneous production of laccase and 90% degradation of
BPA using SSF (solid-state fermentation) process with T. versicolor even
in absence of mediators (Zeng et al., 2017b). SSF process showed better
results in comparison to that of in-vitro conditions with laccase. In
addition to this, laccases are also being explored for treatment of plastic
pollutants (Fujisawa et al., 2001; JMR et al., 2013).
Laccase based bioremediation can also be employed in petroleum
industry as a result of increase in contamination of soil due to petroleum
hydrocarbons like benzene, ethylbenzene, toluene, xylene, etc. Simi­
larly, olive mill extraction leads to generation of large quantity of
phytotoxic substance which can inhibit microbial and plant growth
(Capasso et al., 1992). Liu et al. (2017) reported the removal of petro­
leum hydrocarbons from soil based on sustained release of laccase by
bacteria-white-rot fungi joint remediation, which showed better result
in comparison to the model when either bacteria or fungi was used (Liu
et al., 2017; Zeng et al., 2017a). The effect of decrease in phenolic
content of dry olive mill residue by laccase secreted from Pycnoporus
cinnabarinus and Coriolopsis rigida on tomato plant has been reported
(Aranda et al., 2006).
As discussed earlier, many inhibitors like metal ions are present in
soil which might compete with the compound to be degraded for
binding to the active site of the enzyme. Therefore, results of many
compounds studied in laboratory might show different results when
practically applied on field. Pramanik and Chaudhuri (2018) studied
decolourization of various azo dyes by Podoscypha elegans, a
macro-fungus usually found in soil litter. Laccase from Podoscypha ele­
gans showed maximum utilization of dyes within 21 days of incubation
and also showed activity at higher pH making it suitable for soil biore­
mediation (Pramanik and Chaudhuri, 2018).
The effect of enzymatic treatment of laccase on soil microorganisms
also needs to be considered for its long-term usage in treatment of
contaminated soil. No such significant impact in the plate count of soil
actinomycetes, fungi, or aromatic hydrocarbon degraders when treated
with 10 U/g of laccase for 14 days has been reported (Wu et al., 2008).
Whereas, significant higher bacterial count was reported for the same.
This indicates that laccase has a potential utilization in treatment of soil
contaminated with toxic compounds without damaging the indigenous
microorganisms in the treated soil. However, a recent study has indi­
cated soil ecotoxicity when bioremediation of low-molecular weight
PAHs were carried out in the presence of laccase. This is most likely due
to the structure of the pollutant and the intermediate products other
than quinones formed during the process (Zeng et al., 2021). This sug­
gests careful assessment of the long-term effects before broad-scale
application.
4.2. Bioremediation of pollutants in aquatic environment
The aquatic ecosystem is found to be highly polluted as large amount
13
Journal of Environmental Management 309 (2022) 114676
of toxic contaminants are disposed directly from various industries to
these sites without prior treatment. Phenol compounds are some of the
most common pollutants present in wastewater. Some of these con­
taminants are highly toxic to the organisms/species harbouring in these
sites. Potential of laccase producing microorganism to eliminate pol­
lutants in wastewater is currently one of the most interesting subjects for
researchers in environmental biotechnology. Fig. 3 shows schematic
representation of potential environmental pollutants released from the
industries and their treatment with laccase.
4.2.1. Bioremediation of paper and pulp industry wastewater
Residual lignin is liberated from wood pulp through chloride
bleaching which generates large volume of toxic compounds from paper
and pulp industry. This chloride discharged from paper and pulp in­
dustry is highly coloured, mutagenic and its high concentration might
lead to corrosiveness. Presence of these toxic compounds from these
industry results in production of large volume of organic halogens which
contaminate the environment and can also lead to bioaccumulation
(Christov and Van Driessel, 2003). Moreover, physical and chemical
treatment of these effluents is very expensive. Therefore, efforts are
made to substitute these methods with environment friendly biological
treatment which have proved to be cost-effective and most of these do
not lead to secondary pollution and can reduce biological oxygen de­
mand (BOD) and chemical oxygen demand (COD) to a significant level.
Conventional biological techniques might not effectively remove
chlorolignin compounds. Various cultivation strategies like the mycor
process, continuous flow system, fungal pellets, immobilized culture
treatment, flask and fermenter cultivation can be employed for decol­
ourization of waste water. White-rot fungi secreting laccase have proved
to be effective in their degradation (Christov and Van Driessel, 2003).
Although laccase offers advantage over conventional treatment
methods, finding a stable alkalophilic and thermostable source with
high redox potential is still a challenge. As the pH of effluent from paper
and pulp industry is of alkaline medium, the use of bacterial strain
secreting laccase would be of higher significance. Bioremediation of
paper and pulp mill effluents by using Paenibacillus sp. Strain LD-1
(JX499920) isolated from effluent contaminated soil has been re­
ported (Raj et al., 2014). The results showed significant reduction in
lignin, BOD, COD and phenolic contaminants in comparison to that of
other strains. The addition of mediators in such system may improve the
outcomes but also make the process cost intensive. Ozer et al. (2020)
demonstrated bioremediation in the presence of hydroxycinnamic acid,
a natural mediator that found in lignin and can be released in the
presence of feruloyl esterase (Ozer et al., 2020). More research is needed
in this aspect to find effective natural mediators that will make the
bioremediation process highly cost-effective and eco-friendly.
4.2.2. Bioremediation of pollutants from textile industry
Azo dyes are released in large quantities from textile industries.
These dyes are carcinogenic, toxic, mutagenic and pose threat to the
environment. The toxic compounds present in textile industry effluents
are harmful to aquatic organisms and public health as well (Leechart
et al., 2009). Laccases based techniques were shown to be of great in­
terest in wastewater decolourization and detoxification. The presence of
dye prevents the penetration of sunlight in aquatic ecosystem thereby
reducing the oxygen level. Bacterial degradation or decolourization of
dye effluents often results in the production of colourless dead-end ar­
omatic amines or other metabolites which are generally more toxic than
the parent compounds (Banat et al., 1996; Kulla et al., 1983) and may
therefore have poor adaptability and limited application (Kulla et al.,
1983). Therefore, it is important to develop an effective and
environment-friendly approach for the removal of dye at an acceptable
level and affordable cost. In this context, white-rot fungi secreting lac­
case are one of the most effective class of microorganisms used in the
synthetic dye degradation.
Reports have shown that indigoid dyes got decolourized rapidly in
S.F.F. Zofair et al.
Fig. 4. Potential applications of laccase in different industries.
comparison to azo dyes in a single step procedure with free as well as
immobilized laccase on controlled porosity carrier (CPC) glass beads.
For some dyes, decolourization was greater with free laccase but
detoxification was more effective with immobilized laccase (Champagne
and Ramsay, 2010). The degradation of various dyes including mala­
chite green from extracellular yellow coloured laccase purified from
fermentation broth of white-rot fungus Trametes sp. (Wang et al., 2018a)
has been demonstrated. Trametes versicolor is a good candidate for
decolourization of malachite green, with around 95% decolourization
rate (Qin et al., 2017). Reports have shown that anthraquinone dyes
found in industrial wastewater cannot get degraded by laccase in the
absence of redox mediators because of its chemical structure (Couto and
Herrera, 2007). Hence redox mediators like ABTS or HBT are added to
increase the decolourization rate. Besides this, mediator also helps to
recover the lost enzymatic activity which occurs usually due to the
presence of inhibitor metal ions like Pb+2, Hg+2 and Li+(Xu et al., 2018).
Recently Liu et al. (2020) investigated the synergistic effect of
white-rot fungi microbial fuel cell (WRF-MFC) with improved power
generation and dye decolourization efficiency, and has a great potential
in waste water treatment. The WRF seeding on cathode and the presence
of carbon and copper ions in the solid medium causes considerable in­
crease in laccase activity, thereby enhancing its performance (Liu et al.,
2020).
4.2.3. Bioremediation of pollutants from pharmaceutical industry
Most prevalent compounds found in pharmaceutical waste include
antibiotics, anti-inflammatories, beta-blockers, therapeutic hormones
and analgesics along with their by-product and metabolites which can
be more harmful than the parent compound and may result in
14
Journal of Environmental Management 309 (2022) 114676
bioaccumulation (Tiwari et al., 2017). The adverse effect of these
compounds on aquatic organisms makes the removal or treatment of
these emerging pharmaceuticals pollutants a major concern.
Laccase appears to be a promising alternative for degradation of
toxic effluents from pharmaceutical industries. Studies have shown high
efficiency of laccase in removal of antibiotics, anti-inflammatory anal­
gesics and anti-depressant compounds. Among these, laccase has high
preference for compounds with negative charge containing nitrogen
compounds as seen in many well-known laccase substrates (Taheran
et al., 2017; Tran et al., 2010). Morphine is highly used as an analgesic
and presents a major problem to the environment (Huber et al., 2018).
Morphine elimination has been studied even at high concentration of
6–60 g/L in 180 min by using laccase isolated from Myceliophthora
thermophila in absence of mediators. Even at alkaline pH morphine
removal was achieved but with low oxidation rates (Huber et al., 2018).
The use of mediator enhances the efficiency of degradation of con­
taminants (Kumar and Cabana, 2015). However, the degradation
products should be evaluated and studied for its toxicity.
Time-dependent increase in toxicity when syringaldehyde was used
along with laccase for the removal of antibiotics has been reported, even
though the rate of degradation increased (Becker et al., 2016). However,
this also depends on the mixture of compounds and the type of mediator
involved. An alternative to the use of mediators is the addition of metal
ions along with laccase. Addition of metal ions like Ag+2 and Mn+2 in
optimum amounts enhances the activity of laccases and has shown to
enhance the degradation of tetracycline, quinolones and β-lactam anti­
biotics (Lueangjaroenkit et al., 2019). This method could be a promising
approach for bioremediation of pharmaceutical waste.
S.F.F. Zofair et al.
4.2.4. Bioremediation of pollutants from food industry
Olive oil extraction plants generate olive mill waste (OMW) as a by-
product in quantities higher than 30 million m3 per year. It consists of
high concentration of toxic phenolic components which shows high
ecotoxicity towards aquatic organisms. Therefore, treatment of OMW is
a major issue. Some fungal strains can grow in OMW without additional
supplements and show high laccase activity (Tsioulpas et al., 2002).
However, it was reported that some of the oxidation products obtained
were more toxic in comparison to the parent compound. It has been
demonstrated that OMW treatment showed around 67% and 40%
reduction in phenolic content and colour reduction, respectively, in 24 h
in the presence of HBT (Minussi et al., 2007). The production of laccase
through submerged fermentation of olive-mill by-product and simulta­
neous removal of phenolics present in OMW has been done (Elisashvili
et al., 2018). This method can prove to be very cost-effective and
environment friendly for phenolic reduction in olive mill by-product.
Besides OWM, beer-factory waste also contains high levels of poly­
phenol which needs to be treated before discharge. Laccases are known
to be highly effective in bioremediation of brewery, distillery and soy
sauce waste as well (Wang et al., 2021a, 2021b). Phenolic compounds in
red wine are known to induce laccase production in this strain leading to
high titres of laccase making this process more resourceful (Strong and
Burgess, 2007).
5. Laccase engineering
Even though laccases are widely utilized for different industrial ap­
plications (Fig. 4), the extreme reaction condition often hampers the
activity of laccase or makes it unstable. Engineering of the enzyme might
help to understand the structure-function relationship and overcome
these limitations. Laccases have been subjected to rational, semi-
rational, and directed evolution to enhance its properties and to make
it highly suitable for industrial use (Mate and Alcalde, 2015).
5.1. Rational approach
In this approach, the residues in the substrate binding pocket of
laccase are subjected to site-directed mutagenesis to obtain the recom­
binant enzyme by replacement of the existing residues. It helps to un­
derstand how residues in the vicinity of catalytic site influence the
activity and redox potential of laccase (Pardo and Camarero, 2015).
Site-directed mutagenesis of two hydrophobic residues Leu386 and
Ile494 near T1 site to alanine resulted in mutants showing significant
change in spectral properties of T1 and T2 sites. The mutants had
comparatively low redox potential, which might be due to the increased
solvent accessibility at the binding site (Durão et al., 2008). Subse­
quently, Khodakarami et al. (2018) carried out site-directed mutagen­
esis in laccase form Bacillus HR03. T415G, T418I, T415I and
T415G/T418I variants were produced which are near the T1 site.
Among these variants T415I showed four folds increase in catalytic ef­
ficiency towards ABTS along with 2 folds increase in thermal stability
(Khodakarami et al., 2018b). A common limitation faced in expression
of bacterial laccases is the formation of insoluble protein aggregate­
s/inclusion bodies and low thermostability. Bacterial laccase Lac15-His6
was rationally designed by deleting His-tag which is critical for the
formation of inclusion bodies. Additionally, the flexible residues
323–332 were deleted as they decrease the thermostability. The
resulting Lac15D was thus completely expressed and was also thermo­
stable (Fang et al., 2014).
Computational approach has also been used to rationally design
laccase by predicting the substrate binding of produced variants. Two
active points mutations were introduced in the enzyme by computa­
tional means which enabled efficient electron transfer and hence the
rate of oxidation of aniline resulting in increased kcat by 2 folds (Santiago
et al., 2016). Random mutagenesis in combination with molecular
simulation studies in Fusarium oxysporum Gr2 laccase resulted in new
15
Journal of Environmental Management 309 (2022) 114676
laccase with mutations at Ile52Thr, Arg515Thr and Arg561Thr exhib­
iting 10 folds increase in catalytic efficiency (Kwiatos et al., 2020). In
addition to this, rational designing of the enzyme resulted in its modified
form with large binding pockets which can carry out the oxidation of
bulky PAH seven in the absence of mediators (Chiadò et al., 2021).
For several decades, the molecular basis of laccase and its structure-
function relationship were not clearly understood. The rationally
designed mutants have provided new insights into the role of different
residues and parameters affecting the catalytic activity of the enzyme in
an elaborate manner. These mutants can be further subjected to directed
evolution as they would be more tolerant to the acquisition of new
mutations.
5.2. Semi-rational approach
Prior knowledge on protein structure, sequence and function are
used to prepare combinatorial libraries with mutations targeted towards
the hotspot residues. This is of considerable advantage as it focuses on
areas which are more likely to give positive results. Combinatorial
saturation mutagenesis (CSM) is commonly employed in this approach
to obtained enzyme with increased activity and stability (Kunamneni
et al., 2008; Mate and Alcalde, 2015). Ser510 and Leu513 were targeted
in previously evolved laccase from Myceliophthora thermophila by CSM.
Libraries constructed in S. cerevisiae were screened to obtain mutant 7E1
with 3 folds better activity. 7E1 showed S510G mutation which affects
the C-terminal plug and modulates the redox potential of laccase by
regulating the transit of oxygen and water to the tri-nuclear copper
cluster (Alcalde et al., 2006). Subsequently, when iterative cycles of
CSM were performed on mutant laccase from Ascomycete Mycelioph­
thora thermophile, the results suggested that the tripeptide 509VSG511 had
connection with C-terminal plug (Zumarraga et al., 2008).
Site-saturation mutagenesis was performed on four coordinated po­
sitions of fifth copper in copper efflux oxidase (CueO) from E. coli to
determine the role of fifth copper binding site in CueO electrocatalysis.
The generated library was screened for beneficial variants. The CueO
variants showed improved efficiency of electron transfer as a result of
increase in localized structural stability and decreased distance between
T1 and fifth copper (Zhang et al., 2020a). Similarly, Asp116 mutants
were produced by site-directed mutagenesis to determine its role in the
reduction of O2 to water in CotA laccase. The negative charge on Asp116
was found to be critical in regulating the protonation mechanism of
dioxygen-reduction process and maintaining the local geometry and
water connectivity at the tri-nuclear copper state (Silva et al., 2012).
Such semi-rationally produced mutants will provide a new and effective
strategy to manipulate laccases for better efficiency.
Semi-rational approach based on loop engineering has led to the
production of laccase with improved tolerance to ionic liquids. Engi­
neering laccase loop connecting domain 1 and 2 by substitution of
Glu188 showed remarkable improvement in ionic liquid tolerance and
thermal stability (Dabirmanesh et al., 2015). Similarly, alanine substi­
tution at A285, A310, A312, A318 in loop L1 connecting domain 2 and 3
resulted in loop flexibility and improved activity in the presence of ionic
liquids (Wallraf et al., 2018).
Semi-rational approach is advantageous for modification of laccase
as it can carry out simultaneous mutations and most of the active site
mutations are coupled and show synergistic effects (Chica et al., 2005).
Rational and semi-rational methods involve mutagenesis in the vicinity
of the active site of the enzyme. Whereas, mutation at a site distant from
the active site of the enzyme can be performed in directed evolution in
order to increase its activity.
5.3. Directed evolution
This approach tailors specific traits required for a particular appli­
cation via random mutagenesis or DNA recombination. This method
does not require any in-depth knowledge of structure-function
S.F.F. Zofair et al.
Fig. 5. Factors to consider when using laccases on an industrial scale.
relationship. It involves reducing the evolutionary time scale from mil­
lions of years to few months or weeks in lab (Maté et al., 2011). The
mutations accumulated at active site during evolution, shifts the binding
mode into more buried substrate position. This provides highly
favourable electrostatic environment for the oxidation of substrate and
is beneficial for the overall activity of laccase (Monza et al., 2015).
S. cerevisiae has been used widely for heterologous expression of several
high-redox potential laccase via directed evolution. S. cerevisiae are the
preferred host for directed evolution of fungal laccases due to easy ge­
netic manipulation and high frequency of homologous DNA recombi­
nation with proof-reading activity (Aza et al., 2021; Gonzalez-Perez
et al., 2012). The mutagenic libraries transformed in S. cerevisiae un­
dergo screening for the selection of best variants which are further used
as parental type for subsequent rounds of evolution. Myceliophthora
thermophile subjected to laboratory evolution in S. cerevisiae led to 170
fold and 7.5 fold increase in activity in water and ethanol respectively
(Bulter et al., 2003; Zumárraga et al., 2007). Similarly, Pycnoporus cin­
nabarinus laccase was subjected to directed evolution by heterologous
expression in S. cerevisiae, where its native signal sequence was replaced
by α-factor prepro-leader. It was subjected to six rounds of evolution
which combined mutagenic PCR with in vivo DNA shuffling and back­
crossing recombination, resulting in an overall increase in activity by
8000 folds. An increase in kcat by 13.7 folds was also observed
(Camarero et al., 2012).
Blood tolerant laccase have been developed by this approach to
extend its use in nanobioelectronic devices and medical bioassays. The
laccase variants with F454 P/T/A/G/R/E mutations showed 2.5 fold
increase in catalytic activity in blood-buffer and were not sensitive to
chloride ion concentration (Mate et al., 2013). Directed evolution
approach is also used to develop chimeric laccases with high-redox
potential and combined characteristics (Pardo et al., 2012). Besides
this, computer-aided mutagenesis in combination with directed
16
Journal of Environmental Management 309 (2022) 114676
evolution has been used to design laccase with improved stability with
respect to pH and temperature (De Salas et al., 2019; Mateljak et al.,
2019).
Engineering of laccases needs to be performed, keeping in mind the
substrate to be utilized as the substrate binding pocket differs for
different laccases which is responsible for different oxidation capacity
(Pardo et al., 2016). Sometimes directed evolutionary approach leads to
the loss of the beneficial mutations when one parameter is chosen over
the other during the screening process. In relation to this, direct evo­
lution followed by rational design can be used to restore the lost bene­
ficial mutations resulting in highly stable and active enzyme (Sayut and
Sun, 2010). As direct deduction of structural determinant of protein is
quite difficult, directed evolutionary approach seems to be an extremely
promising alternative for designing laccases. This approach might help
laccase to adapt to extreme conditions, facilitating its use in different
industrial purpose.
6. Conclusion
Laccases exhibits broad range of substrate specificity and have
different substrate preferences; hence more studies need to be done to
elucidate the structure of the catalytic site of laccases from different
sources to understand their differential interaction with the substrates.
Some compounds require the use of mediators in laccase catalyzed re­
action which leads to enhanced degradation rate. Most of the mediators
used with the enzyme are artificial, costly and sometimes lead to sec­
ondary waste generation. Hence, more effective natural mediators
should be searched in order to cut down the cost of the process. Alter­
natively, co-immobilization of laccase and mediator for the reaction
system might be feasible for industrial application and allow the reuse of
mediators as well, thereby eliminating secondary pollution. The
immobilization of laccase can be further controlled through site-directed
S.F.F. Zofair et al.
mutagenesis which has reported to show an enhancement in enzymatic
activity. More understanding of interaction between laccase and the
support might help in exploring new possibilities of improving the
enzyme activity. This review provides a comprehensive summary of
most factors to consider while working with laccases in an industrial
setting (Fig. 5).
Laccase based methods face limitations at an industrial scale due to
cost involved in production, cost of mediators and its toxicity, alkaline
conditions, operational stability and presence of various inhibitors in the
effluents. The availability of robust laccases which are highly stable/
tolerant in operational conditions is one of the main bottlenecks for its
practical application. Research on protein engineering or genetic
manipulation of laccase will help in stabilizing and improving the per­
formance of the enzyme. Tailoring of laccases according to the type of
substrate to improve their catalytic performance is the key challenge
faced by various researchers. Future approach on engineering of laccases
with high redox potential and ability to withstand alkaline conditions in
the presence of inhibitors can be done via mutagenesis and proper
screening of the mutants by computational simulation. These engi­
neered enzymes combined with suitable support are expected be a
promising alternative compatible for industrial use especially for the
purpose of bioremediation.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The authors thankfully acknowledge the Interdisciplinary Biotech­
nology Unit (IBU) of the Aligarh Muslim University for providing
research facilities. S.H.K. acknowledges Indian Council of Medical
Research, Govt. of India for providing financial assistance in the form of
senior research fellowship.
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