Archives of

1. Introduction
In general, enzymes are very important alternatives for many industrial applications due to their high specificity, low investment costs, low operating energy requirements, and natural origin. In particular, enzymes produced by white-rot fungi have become powerful tools in research related to waste treatment, environmental protection, degraded area restoration, and industrial wood processing. The use of enzymes in industrial processes can reduce or replace the use of aggressive compounds and allow processes at milder temperatures and pH values [1].
Laccase (EC 1.10.3.2, p-diphenol oxidase) has been studied since the 19th century. Laccase was discovered by Yoshida in the exudate of the lacquer tree Rhus vernicifera in 1883. However, Bertrand and Laborde demonstrated in 1896 that laccase is a fungal enzyme [2,3]. Laccases are copper-containing enzymes that catalyze the oxidation of various organic and inorganic substrates, such as monophenols, diphenols, polyphenols, aminophenols, methoxyphenols, aromatic amines, and ascorbate, with the concomitant four-electron reduction of oxygen to water [4]. Laccase is a member of the large blue copper proteins or blue copper oxidases. Other enzymes in this group include ascorbate oxidase in plants and the plasma protein ceruloplasmin in mammals. The ability of laccases to oxidize phenolic compounds and reduce molecular oxygen to water has led to intensive research on these enzymes [2].
Laccase enzymes are found primarily in fungi. They are also found in various organisms, including plants, bacteria, and a few insects [5,6]. Fungal laccase enzymes (e.g., laccase enzyme from the
edible mushroom Lentinus edodes) are frequently used in the paper industry and are studied more frequently than other organisms due to their high ability to degrade wood [7,8]. Due to its high stability and specificity, it can be used in various industries and biotechnological applications to remove environmental pollutants such as antibiotics. The presence of these pollutants in wastewater leads to contamination of natural water sources [9]. Table 1 shows the general uses of laccase enzyme and its sources.
2. Chemical structure and properties of laccase
Laccase enzymes are glycoproteins with enormous diversity in mass, carbohydrate composition, and the number of protein chains. Most of the studied laccase enzymes of fungal origin have monomer, dimer, or
tetramer structures. The carbohydrate chain may be
associated with the stability of enzymes [7]. The molecular
weight of monomeric glycoproteins differs from 50 to
130 kDa. The carbohydrate moiety consists of mannose,
acetylglucosamine, and galactose, which make up 10%-
45% of the total mass of a monomeric enzyme [11].
2 .1. Thermal studies on activity and stability of laccase
Laccase enzymes can show different stability and optimal
activity depending on their substrate, pH condition, and
temperature. It is reported that their optimal activity even
changes in the presence and absence of light. A study has
shown that laccase produced by Tricholoma matsutake
reaches its optimal activity at 30 °C in the light; however,
this temperature drops to 25 °C when the incubated
fungus grows in the dark [12]. More research has recently
been focused on the techniques that increase the stability
and activity of laccase enzymes in higher temperatures.
In 2020, Wang et al found that laccase immobilized
onto zeolite imidazolate framework-67 (ZIF-67)
indicates remarkable stability and higher reusability
[13]. Moreover, pre-incubation of the laccases, extracted
from Marasmius quercophilus, at 40 °C and 50 °C has
been shown to increase the stability [7]. In general, the
optimal temperature for the activity of laccase enzymes
ranges from 30 °C to 50 °C [11], and pre-incubation
and utilization of metal frameworks can help with the
improvement of the enzyme.
2.2. Impacts of different pH conditions on laccase
It is believed that the initial pH for laccase enzyme
production ranges from 4.5 to 6.0 [12]. This variation
originates from the redox potential differences between
the phenolic substrates and the copper oxidation sites,
meaning that the enzyme works better at higher pH values
with a difference in redox potential between the substrate
and copper atom in the active site. Besides, the hydroxide,
produced from the reaction, binds to the other 3 active
sites, which results in the inhibition of the enzyme [7].
3. Sources and occurrence
According to the BRENDA database, more than 300
fungal laccases have been discovered that are responsible
for fungi sporulation and formation of the fruit body,
degradation of lignin, pigment production, and defense
against stress [14]. The most famous species of these
fungi, known for its ability to degrade lignin, is white-rot
basidiomycete [7]. Pycnoporus cinnabarinus, Pycnoporus
sanguineus, and Neurospora crassa have been reported
to produce laccase [7]. Fungal laccases can be produced
extracellularly, intracellularly, or even both, depending
on fungal species.
As already mentioned, plant laccase was first extracted
from Rhusvernicifera tree. Recently, Gossypium spp.
[15], Oryza sativa [16], Prunus avium [17], Pyrus
bretschneideri [18], Amborella trichopoda, Glycine max,
Physcomitrella patens, Ricinus communis, Triticum
aestivum, Vitis vinifera [16], Setaria viridis [19], and
Zea mays [20] have been found to have genes to encode
laccase enzyme [14]. Plant laccases undertake numerous
physiological and biochemical actions such as lignin
polymerization, defense mechanism, wound healing,
maintenance of the structure, and the polymerization of
phenolic compounds [14]. Although there has not been
much research on plant-derived laccase enzymes, some
studies have suggested that these kinds of laccase enzymes
are good candidates for remodeling and improving the
production of biofuels [14].
Laccase enzymes do also exist in some species of
bacteria such as Azospirillum lipoferum [7], Bacillus, and
Streptomyces genus [14]. Bacterial laccases have been
shown to play some important biological roles, including
pigmentation against UV light, degradation of lignin, and
creation of antibiotics [14].
3.1. Fungal laccase
Fungal laccases are of great importance due to their
widespread applications in agriculture, medicine, textiles,
and the pulp industry. In Table 1, some fungal laccases
with their specific applications in the industry are
presented [10]. Ameri et al showed that by optimizing
the growth conditions of mycelium and polysaccharides
of Lentinus edodes on walnut shell by-products using
response surface analysis, polysaccharides of Lentinus
edodes can biologically decompose polyaromatic
hydrocarbon by producing laccase enzyme [21].
4. The importance of enzymes
In the last 2 decades, climate change and human impacts
on nature have urged scientists and researchers to
innovate or discover bio-friendly solutions to reduce
or remove industrial damage to the environment. It is
considered one of the green enzymes to be replaced with
various harmful industrial processes. Additionally, its
Table 1. Production Sources and Uses of Laccase [10]
Plant Insects Bacteria Fungi
Source of laccase
enzyme
Lacquer, Mango,
Mung, Been,
Sycamore
Bombyx, Calliphora, Diploptera, Drosophila, Lucillia,
Manduca, Musca, Orycetes, Papilio, Phormia,
Rhodnius, Sarcophagi, Schistocerca, Tenebrio
Azospirillum lipoferum, Marinomosas
mediterranea, Streptomyces griseus,
Bacillus subtillis
Ascomycetes,
Basidiomycetes,
Deuteromycetes
Application
Biosensor, immunochemical assays, biodegradation of wastes, organic compound, gold nanoparticle synthesis, DNA labeling, baking
industry
Arch Hyg Sci. Volume 12, Number 4, 2023 209
A brief review on laccase enzyme by lentinus edodes and its applications
thermostability, versatility, and biocatalyst features have
allowed laccase to be widely utilized in the paper and pulp
industry, food processing industry, and even medical
approaches and treatments [11].
5. Applications
5.1. Removal and degradation of antibiotics
One of the major global concerns of the current era
is antimicrobial resistance. According to WHO, there
are several reasons behind antimicrobial resistance
including improper consumption of antibiotics by
patients, overprescribing of antibiotics, overuse of
antibiotics in livestock, and especially lack of access to
clean and sanitized water. Unfortunately, the presence
of antibiotics in wastewater of hotspots, including
hospitals, wastewater treatment plants, animal feeding
operations, and aquaculture operations, has accelerated
the antimicrobial resistance [22]. These municipal sewage
treatment plants and industrial wastewater treatment
plants are major sources of antibiotic-resistance genes
[23]. Therefore, there has been extensive research on
the removal of antibiotics from the WWTP sewage. As
mentioned earlier, laccase can also degrade nonphenolic
compounds, including some sorts of antibiotics. As
a result, it is proved that laccase can be a successful
solution for clearing wastewater of treatment plants from
antibiotics. Prieto et al showed that laccase produced by
Trametes versicolor [8] could degrade more than 90%
of ciprofloxacin and norfloxacin, which is in line with
the results of the study by Becker et al [24]. However, it
should be considered that intermediaries are essential for
the oxidation of other nonphenolic substrates of laccase
to take place.
5.2. Elimination of antibiotics by laccase and
syringaldehyde
Becker et al reported that a combination of immobilized
laccase (Trametes versicolor) and SA mediator in an
enzymatic membrane reactor eliminated a mixture of 38
antibiotics at an environmentally relevant concentration
(10 μg·L−1) [24]. SA is the main mediator, mostly occurring
in plants, that helps with the degradation of lignin. This
organic compound has lower redox potential compared
to laccase. Therefore, when it comes to the reaction, SA
gets oxidized faster and the reaction releases radicals that
oxidize nonphenolic compounds, which is exactly how
mediators help laccase with degrading other nonphenolic
substrates.
Becker et al showed that the addition of SA enhances the
degradation of antibiotics. Of the 38 antibiotics, 32 were
degraded by more than 50% within 24 hours. Amoxicillin
and ampicillin had the highest removal percentage (up
to 90%) among the other penicillins, which were mostly
stable. The treatment had no significant impact on the
removal of quinolones, metronidazole, and trimethoprim
(less than 30%). However, 60% of pipemidic acid was
removed [24].
Degradation of antibiotics was intensified at higher
concentrations of SA (1000 μmol·L-1). Incredibly, 17 out of
38 antibiotics were cleared more than 90% after 24 hours.
Sulfonamides, except for sulfanitran, were observed to
have the highest removal rate ( > 97% removal after 2
hours). Moreover, the degradation of quinolones was
improved, except for cinoxacin (15% removal), reaching
more than 70% after 24 hours. Fluoroquinolones, except
for difloxacin (49%), orbifloxacin (33%), and flumequine
(42%), also showed the same pattern of removal.
However, SYR1000 could not enhance the removal of all
38 antibiotics. Comparing the removal of tetracyclines
with SYR1000 to its lower concentration, it was seen that
this class of antibiotics indicated better decomposition
(60%–90% after 24 hours) with SYR1000 [24].
In conclusion, 32 out of 38 antibiotics at environmentally
relevant concentrations can be cleared (more than 50%)
in an enzymatic membrane reactor containing laccase
immobilized on ceramic membranes and SA. Whereas
there was much removal in the experiment without SA
as a mediator [24].
5.3. Laccase treatment in the presence of HBT compared
to manganese peroxidase
Many studies have emphasized the importance of the
accompaniment of mediators to laccase enzymes in
accelerating the catalyzation of antibiotics. Not only
SA but also many other chemical compounds like
hydroxybenzotriazole (HBT) [25] can boost oxidation
reactions with laccase enzymes. It has been reported
that the laccase–HBT system could clear tetracycline,
chlortetracycline, doxycycline, and oxytetracycline from
reaction mixtures [25]. Furthermore, it has been reported
that ligninolytic enzymes (manganese peroxidase, lignin
peroxidase, and laccase) can degrade antibiotics [26].
Manganese peroxidase is an organic compound that
is produced by white rot fungi, especially Trametes
versicolor. It consists of a heme that functions as its
peroxidase site, which converts Mn (II) to Mn (III) [26].
Suda et al observed that laccase–HBT system was more
effective in the degradation of antibiotics than manganese
peroxidase and laccase alone [25]. In addition, the removal
rates of tetracycline, chlortetracycline, doxycycline, and
oxytetracycline were increased by 16%, 48%, 34%, and
14%, respectively [24].
6. Biotechnological purposes
One of the characteristics of laccase enzymes is that they
can catalyze substrate, needing high reduction potential
to get oxidized. Furthermore, laccases produced by
white-rot basidiomycete fungi have shown a high redox
potential and the ability to remove Bisphenol A in
applications associated with bioremediation [14]. Laccase
Ameri Shah Reza et al
210 Arch Hyg Sci. Volume 12, Number 4, 2023
enzyme has a high ability to catalyze various biological
compounds and can remove complex structures such as
antibiotics in the bioremediation process.
6.1. Potential for the decolorization of textile dyes
Due to their oxidative characteristics, laccases have
captured the attention of manufacturers as they can be
applied in industrial processes such as decolorization of
textile dyes, Malachite Green decolorization, and fiber
confining [12].
6.2. Medical treatments
Pathogenic yeast Cryptococcus neoformans encodes
the genes of laccase, which plays an important role
in the pigmentation of melanin and the production
of immunomodulatory agents. Therefore, it can be
considered a remarkable virulence [12]. Moreover,
laccase extracted from oyster mushroom (Pleurotus
ostreatus) is used as herbal medicine to inhibit hepatitis
C virus replication [12].
7. Conclusion
Laccase is one of the leading future bioremediation
solutions for the degradation of industrial pollutants
and antibiotics. One of the chief functions of the laccase
enzyme is the removal of antibiotic pollutants, which
have become one of the biggest challenges of this decade.
Maintaining the stability of laccase at high temperatures
in renewable industrial processes such as textiles, pulp
industry, medical treatments, and bioremediation
processes is a great challenge that scientists are facing.
Despite the decontamination ability, laccase treatment
reactions can generate some other pollutants. Recent
studies have been working out solutions to minimize the
production of these pollutants.
The elimination of antibiotic pollutants in relevant
environmental conditions using laccase and SRY as a
mediator is a significant breakthrough.
Authors’ Contribution
Conceptualization: Mahdieh Ameri Shah Reza.
Data curation: Mahdieh Ameri Shah Reza, Ali Darvish.
Formal analysis: Mahdieh Ameri Shah Reza, Ali Darvish.
Funding acquisition: Mahdieh Ameri Shah Reza.
Investigation: Mahdieh Ameri Shah Reza, Ali Darvish.
Methodology: Mahdieh Ameri Shah Reza, Alireza Rasouli, Ali
Darvish.
Project administration: Mahdieh Ameri Shah Reza.
Resources: Mahdieh Ameri Shah Reza-Alireza Rasouli, Ali Darvish.
Software: Mahdieh Ameri Shah Reza-Alireza Rasouli, Ali Darvish.
Supervision: Mahdieh Ameri Shah Reza, Alireza Rasouli.
Validation: Mahdieh Ameri Shah Reza, Alireza Rasouli.
Visualization: Mahdieh Ameri Shah Reza, Alireza Rasouli, Ali
Darvish.
Writing–original draft: Mahdieh Ameri Shah Reza, Alireza
Rasouli, Ali Darvish.
Writing–review & editing: Mahdieh Ameri Shah Reza, Alireza
Rasouli, Ali Darvish.
Competing Interests
The authors declared no conflict of interest.
Funding
This work was funded by a grant (grant no: IR.MUQ.REC.1401.076)
from the Vice for Research and Technology of Qom University of
Medical Sciences, Qom, Iran (Project research code: 14011563).
References
1. Guimarães LR, Woiciechowski AL, Karp SG, Coral JD,
Zandoná Filho A, Soccol CR. Laccases. In: Pandey A, Negi S,
Soccol CR, eds. Current Developments in Biotechnology and
Bioengineering. Elsevier; 2017. p. 199-216. doi: 10.1016/
b978-0-444-63662-1.00009-9.
2. Thurston CF. The structure and function of fungal laccases.
Microbiology. 1994;140(1):19-26. doi: 10.1099/13500872-
140-1-19.
3. Levine W. Laccase, a review. In: The Biochemistry of Copper.
New York: Academic Press Inc; 1965. p. 371-85.
4. Galhaup C, Goller S, Peterbauer CK, Strauss J, Haltrich D.
Characterization of the major laccase isoenzyme from
Trametes pubescens and regulation of its synthesis by metal
ions. Microbiology (Reading). 2002;148(Pt 7):2159-69. doi:
10.1099/00221287-148-7-2159.
5. Mayer AM, Staples RC. Laccase: new functions for an old
enzyme. Phytochemistry. 2002;60(6):551-65. doi: 10.1016/
s0031-9422(02)00171-1.
6. Guest TC, Rashid S. Anticancer laccases: a review. J Clin Exp
Oncol. 2016;5(1):1-7. doi: 10.4172/2324-9110.1000153.
7. Kunamneni A, Ballesteros A, Plou FJ, Alcalde M. Fungal
laccase—a versatile enzyme for biotechnological applications.
In: Méndez Vilas, ed. Communicating Current Research and
Educational Topics and Trends in Applied Microbiology. Vol
1. Badajoz: FORMATEX; 2007. p. 233-45.
8. Prieto A, Möder M, Rodil R, Adrian L, Marco-Urrea E.
Degradation of the antibiotics norfloxacin and ciprofloxacin
by a white-rot fungus and identification of degradation
products. Bioresour Technol. 2011;102(23):10987-95. doi:
10.1016/j.biortech.2011.08.055.
9. Batt AL, Aga DS. Simultaneous analysis of multiple classes
of antibiotics by ion trap LC/MS/MS for assessing surface
water and groundwater contamination. Anal Chem.
2005;77(9):2940-7. doi: 10.1021/ac048512 + .
10. Senthivelan T, Kanagaraj J, Panda RC. Recent trends in
fungal laccase for various industrial applications: an ecofriendly
approach - a review. Biotechnol Bioprocess Eng.
2016;21(1):19-38. doi: 10.1007/s12257-015-0278-7.
11. Singh D, Gupta N. Microbial Laccase: a robust enzyme and
its industrial applications. Biologia. 2020;75(8):1183-93. doi:
10.2478/s11756-019-00414-9.
12. Dana M, Bakhshi Khaniki G, Mokhtarieh AA, Davarpanah
SJ. Biotechnological and industrial applications of laccase: a
review. J Appl Biotechnol Rep. 2017;4(4):675-9.
13. Wang Z, Ren D, Yu H, Jiang S, Zhang S, Zhang X. Study
on improving the stability of adsorption-encapsulation
immobilized Laccase@ZIF-67. Biotechnol Rep (Amst).
2020;28:e00553. doi: 10.1016/j.btre.2020.e00553.
14. Brugnari T, Braga DM, dos Santos CSA, Torres BH, Modkovski
TA, Haminiuk CW, et al. Laccases as green and versatile
biocatalysts: from lab to enzyme market—an overview.
Bioresour Bioprocess. 2021;8(1):131. doi: 10.1186/s40643-
021-00484-1.
15. Balasubramanian VK, Rai KM, Thu SW, Hii MM, Mendu
V. Genome-wide identification of multifunctional laccase
gene family in cotton (Gossypium spp.); expression and
biochemical analysis during fiber development. Sci Rep.