International Journal of Biological Macromolecules 181 (2021) 683–696

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Exploring current tendencies in techniques and materials for

immobilization of laccases – A review
Lynette Alvarado-Ramírez a, Magdalena Rostro-Alanis a,⁎, José Rodríguez-Rodríguez a, Carlos Castillo-Zacarías a,
Juan Eduardo Sosa-Hernández a, Damià Barceló b,c,d, Hafiz M.N. Iqbal a,⁎, Roberto Parra-Saldívar a,⁎
a
Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey 64849, Mexico
b
Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona, 18-26, 08034 Barcelona, Spain
c
Catalan Institute for Water Research (ICRA-CERCA), Parc Científic i Tecnològic de la Universitat de Girona, c/Emili Grahit, 101, Edifici H2O, 17003 Girona, Spain
d
College of Environmental and Resources Sciences, Zhejiang A&F University, Hangzhou 311300, China

a r t i c l e i n f o a b s t r a c t

Article history: Nanotechnology has transformed the science behind many biotechnological sectors, and applied bio-catalysis is
Received 28 January 2021 not the exception. In 2017, the enzyme industry was valued at more than 7 billion USD and projected to 10.5 bil-
Received in revised form 16 February 2021 lion by 2024. The laccase enzyme is an oxidoreductase capable of oxidizing phenolic and non-phenolic com-
Accepted 26 March 2021
pounds that have been considered an essential tool in the fields currently known as white biotechnology and
Available online 30 March 2021
green chemistry. Laccase is one of the most robust biocatalysts due to its wide applications in different environ-
Keywords:
mental processes such as detecting and treating chemical pollutants and dyes and pharmaceutical removal. How-
Immobilization ever, these biocatalytic processes are usually limited by the lack of stability of the enzyme, the half-life time, and
Laccases the application feasibility at an industrial scale. Physical or chemical approaches have performed different
Bio-catalysis laccase's immobilization methods to improve its catalytic properties and reuse. Emerging technologies have
Nanocarriers been proven to reduce the manufacturing process cost and increase application feasibility while looking for eco-
Novel techniques logical and economical materials that can be used as support. Therefore, this review discusses the trends of en-
Biological macromolecules zyme immobilization recently studied, analyzing biomaterials and agro-industrial waste used for that
intention, their advantages, and disadvantages. Finally, the work also highlights the performance obtained
with these materials and current challenges and potential alternatives.
© 2021 Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
2. Novel techniques for immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
2.1. Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
2.2. Electrospraying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
2.3. Matrix-assisted pulsed laser evaporation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2.4. Soft plasma polymerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2.5. 3D printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
2.6. Laser printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
2.7. Metal-organic frameworks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
2.8. Crosslinked enzyme aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
2.9. Hybrid nanoflowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
3. Novel carriers for immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
3.1. Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
3.2. Agro-industrial waste materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

⁎ Corresponding authors.
E-mail addresses: a00814259@itesm.mx (L. Alvarado-Ramírez), magda.rostro@tec.mx (M. Rostro-Alanis), jrr@tec.mx (J. Rodríguez-Rodríguez), carloscastilloz@tec.mx
(C. Castillo-Zacarías), eduardo.sosa@tec.mx (J.E. Sosa-Hernández), dbcqam@cid.csic.es (D. Barceló), hafiz.iqbal@tec.mx (H.M.N. Iqbal), r.parra@tec.mx (R. Parra-Saldívar).

https://doi.org/10.1016/j.ijbiomac.2021.03.175
0141-8130/© 2021 Published by Elsevier B.V.
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al. International Journal of Biological Macromolecules 181 (2021) 683–696

Declaration of competing interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

1. Introduction disadvantages, are shown in Fig. 1. The simplest methods are adsorption
and entrapment, highlighting that the native structure of the enzyme is
Nowadays, biocatalysts have been considered a strengthened tech- not damaged [8,12]. However, entrapment enzymes have mass transfer
nology in the green chemistry field since the value of enzymes indus- limitations and adsorption loss of enzymes in the operation process
trial sector was projected to 6.3 USD billions for 2021 [1,2]. The use of because of their weak interactions [13–15]. In covalent bonding and
enzymes permits a remarkable selectivity under mild conditions, low crosslinking, conformational changes in enzymatic structure and loss of
energy input, and non-toxic technology. Laccase (benzenediol: oxidore- activity can decrease enzyme activity. Despite this, covalent bonding is
ductase) is a multi‑copper oxidase that combines the four-electron re- the most used method due to the high interactions between the enzyme
duction of dioxygen to water with the one-electron oxidation of four and the support [16–18]. Recent strategies on immobilization have been
substrate molecules [3]. Laccases are monomeric glycoproteins contain- developed using new technologies like jet printing, electrospinning, 3D-
ing about 500 amino acids with a molecular weight of 60–80 kDa and printing, among improving the efficiency of the process and simplifying
carbohydrate content up to 25%. The active site contains four copper the immobilization steps [19–23]. Table 1 lists the techniques used to im-
atoms classified according to their spectroscopic and paramagnetic mobilize laccases and the retained activity after the procedure.
properties in three redox groups: type-1 (T1), type-2 (T2), and type-3
(T3). T1 cooper has the highest redox potential (E°) and is the substrate
2.1. Electrospinning
oxidation site. This ion has two conserved histidines and one cysteine as
equatorial ligands. T2 and T3 form a trinuclear cluster, where the molec-
Electrospinning is a technique mostly used to prepare fibers and
ular oxygen is reduced, and water is released [4–6]. Because they only
membranes in a faster way. This technique produces continuous fibers
require oxygen molecules as reactants and only produce water mole-
with diameters between the micrometer and nanometer range [24,25].
cules as byproducts, thus, named “green catalyst” [4]. Laccases are pro-
Fig. 2 illustrates an electrohydrodynamic process, where a polymer solu-
duced by fungi, plants, bacteria, or insects. It is also considered a
tion can be spun by applying an electric field to obtain the fibers [22].
promising enzyme due to its remarkable catalytic properties as wide
Electrospun fibers have many advantages, such as small diameters, po-
substrate range used by it [4]. This catalytic capacity opens the possibil-
rous structure, high surface area, and superior mechanical properties
ity to use this enzyme for a wide range of industrial applications. Re-
[26–28]. Therefore, it can be applied in tissue engineering, biomedicine,
cently, laccase has been centered on the degradation of xenobiotic
food engineering, biotechnology, and the detection and removal of pollut-
compounds, including dyes and pharmaceutical molecules, and in the
ants. This immobilization process can be done after the fibers are made by
development of biosensors used in the environmental and food indus-
a covalent or entrapment method that successfully immobilizes enzymes
try. However, despite all the advantages of using laccases, some disad-
and shows high stability [29]. Among the different polymers used to im-
vantages include lack of long-term operation, stability, or inability to
mobilize laccase, nylon, chitosan, polyvinyl alcohol (PVA) fibers have
recover the enzyme, make it impossible to use laccase at an industrial
been used. The nature of the components used opens the possibility to
scale [7,8]. The immobilization strategy offers considerable potentiali-
use these fibers for food applications. Furthermore, the enzyme activity
ties that can be used to solve these problems, i.e., reusing enzymes in
was increased after the immobilization process [24,26]. Immobilized
different cycles with increased stability and enzymatic activity [6,7]. En-
laccase by electrospinning, which is recognized as an in-situ method,
zyme immobilization can be performed by a physical or chemical
can be applied for biosensing. For instance, laccases encapsulation in
method. In both cases, the enzyme is retained in a confined space or
PVA with gold nanoparticles to enhance the biosensor's conductivity
linked to a support. It is essential to know the protein characteristics
have been performed [30]. Similarly, Poly (D, L-lactide) (PDLLA) has
for an efficient immobilization and see if the procedure would affect
been used to immobilize laccases by electrospinning. The activity of
the enzyme's performance. However, it has been challenging to estab-
laccase increased by 67%. The increment of activity in these electrospun
lish the optimum immobilization method and support for any enzyme.
fibers could be for the core-shell structure, which gives protection to en-
The immobilization method is influenced by the different chemical
zymes. However, laccase retained activity in two weeks decreased by al-
properties and structures of enzymes [9]. Nowadays, various techniques
most 50% [28].
for immobilization have been in practice and new technologies that
Comparison between the yield obtained by immobilization in nano-
allow more efficient immobilization of enzymes are continuously devel-
fibers by covalent bonding and by encapsulation was performed by
oped [10].
Zdarta et al. [31]. In this research, electrospun nanofibers using poly
To achieve some of the sustainable development goals (SDGs)
(methyl methacrylate) and magnetite nanoparticles were produced.
launched by the United Nations in 2012, proper exploitation of numer-
The nanofibers were first modified for covalent bonding and encapsula-
ous materials in a wide range of areas is desirable [11]. This trend has
tion of laccase. A yield of 100% for encapsulation and 79% for covalent
impacted the enzyme immobilization area, which also represents an en-
bonding were obtained during the immobilization. However, for tetra-
vironmental benefit. Herein, recent materials and enzyme immobiliza-
cycline removal, 100% was removed with free and covalently bonded
tion techniques are reviewed with suitable examples. The work also
laccase, whereas 94% with encapsulated laccase [31]. This performance
highlights the new tendency to use biomaterials and agro-waste mate-
could be for the accessibility to enzyme active sites. Nevertheless, it was
rials for enzyme immobilization.
possible to use both immobilized systems for five cycles. For this reason,
it was possible to form stable catalytic systems for biotransformation of
pollutants or biosensor applications.
2. Novel techniques for immobilization

Physical or chemical methods are the most used immobilization strat-
egies. The five basic immobilization techniques, i.e., (1) adsorption, (2) en-
capsulation, (3) covalent bonding, (4) entrapment, and (5) crosslinking,
are still used in the new technologies. A schematic illustration of five
basic immobilization techniques, along with their advantages and
2.2. Electrospraying
Electrospraying is considered a technology related to electrospinning
because, in both technologies, an application of a high potential electric
field is needed. A polymeric solution is sprayed to obtain particles

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L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
Fig. 1. Schematic illustration of five basic immobilization techniques along with their advantages (green) and disadvantages (red).
(Fig. 3). The process consists of a gentle ionization method that does not
break or disarrange the molecules [26]. Thereby, electrospraying is ex-
tensively used in larges molecules such as enzymes and proteins to
bring them as intact and isolated units in the gas phase at ambient pres-
sure to deposit them in films and microarrays [19]. At a low concentra-
tion, the molecule passes through a small capillary held a few kV high
voltage to the counter electrode. The charges analyte molecules repel
each other and expand the solution/gas interface, the surface tension
Table 1
Comparison of techniques for enzyme immobilization.
Technique Advantages
Electrospray Gentle method, efficient encapsulation, reduce denaturalization. Production of smaller
particles.
MAPLE A simple, cheap, and versatile method
SPP Not use toxic solvents, can be done at atmospheric pressure and room temperature.
Electrospinning A fast and simple method.
Fibers can have different diameters.
Electrospun nanofibers showed longer stability, lifetime
The high surface area and porosity help to the diffusio
3D-printing Low cost of equipment and materials, the design can be easily changed.
It is possible to construct complex internal structures.
Laser printing Enables large-scale manufacturing.
It is possible to use a wide range of inks and printing in different materials.
MOFs Stability and reusability of enzymes have been enhanced
Easy recycling
CLEAs A simple method, low cost, easy to recover
hNFs Higher catalytic activities and stability are obtained. High surface,
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International Journal of Biological Macromolecules 181 (2021) 683–696
of the liquid balances the electrostatic repulsive forces. After this, the
surface tension cannot stand anymore, a spray of charged droplets is
generated, whose size is decreased when the solvent is evaporated
[26]. Castrovilli et al. [19] used this technique to immobilize laccases
on carbon black-nanomodified to fabricate a biosensor for phenolic
compounds. Due to the softness of the process, the enzyme maintained
100% of its activity after immobilization, and it was possible to use the
biosensor for 25 consecutive measurements retaining 100% of its
Disadvantages
There are significant challenges to control the process at larger
scale.
Laser-matter interaction can damage the enzyme.
Enzyme activity is decreased.
Low deposition rates, polymerized coatings have low resistance.
Difficult to make a large volume of scaffold. Use of toxic solvents.
Limited materials, post processing, restricted build size.
High production cost.
Reaction conditions are relatively severe.
Enzyme activity loss is large
Possible denaturalization
Structural features and dimensions are difficult to control.
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
Fig. 2. Immobilization of laccase by electrospinning technique (a) in-situ, (b) after synthesis.
activity, and after 90 days, the enzyme retained 100% of activity. There-
fore, immobilization using this technique offers a good option, and it can
be improved by using other materials.
2.3. Matrix-assisted pulsed laser evaporation
Laser deposition techniques such as Matrix-Assisted Pulsed Laser
Evaporation (MAPLE) can be considered an alternative for enzyme im-
mobilization. This method is considered a simple, cheap, and versatile
method for the deposition of thin films, even to obtain biological thin
films [32,33]. Firstly, the molecule of interest and the solvent must be
frozen. Secondly, the solution and the solvent are vaporized by a laser
impact allowing the molecules of interest to be transferred and depos-
ited as a thin film on support (Fig. 4). Laccase has been immobilized
by this technique using water as a solvent and then deposited onto
the glass. Although no substantial modification was observed during
the deposition process, only 10% of the enzyme was active after the
process [32]. Verrastro et al. [33] used benzene instead of wáter, as a
solvent, for the target preparation, allowing less pulse energy to gener-
ate laccase thin films. Also, the reproducibility of the deposition of
MAPLE was coefficient of variation 12.9%, and the biosensor showed
working stability of 5 measurements and stability for 100 days at 4 °C.
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International Journal of Biological Macromolecules 181 (2021) 683–696
Moreover, MAPLE was considering a promising technique for laccase-
based biosensor's production at mass scale.
2.4. Soft plasma polymerization
Soft plasma polymerization (SPP) is a technique used for deposition
coatings with the advantage that it uses non-toxic solvents at atmo-
spheric pressure and room temperature. These conditions make the de-
position of organic molecules without denaturalization and open the
possibility of working with enzymes. In this technique, the molecule
or the organic compound is deposited as a crosslinked thin film on a sur-
face. Chemical precursors, such as TMCTS (tetramethylcyclosiloxane),
HMDSO (hexamethyldisiloxane), HMDSN (hexamethyldisilazane), TEOS
(tetraethoxysilane), and TMDSO (tetramethylodisiloxane) can be used.
They interact with the organic compound making a crosslinking network
between them. However, it is essential to remark that the energy density
of the plasma discharge, carrier gas, and the solid used as a substrate can
affect the polymeric coatings' properties [21]. Also, SPP has been used to
construct a laccase-based biosensor to determine dopamine [34]. The bio-
sensor was constructed with a voltage of 3 kV (30 s time deposition), 10 L/
min for helium flow rate, and 200 μL/min for laccase solution flow rate;
presenting two linear ranges (0.1 μmol/dm3 to 10 μmol/dm3 and
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
Fig. 3. Immobilization of laccase by electrospray technique.
10 μmol/dm3 to 50 μmol/dm3) with remarkable dopamine detection sen-
sitivities (3.63 μA∗dm3/μmol and 1.33 μA∗dm3/μmol). Another example of
the construction of a laccase-based biosensor by the SPP technique was
developed by Malinowski et al. [35] to detect dihydroxybenzene iso-
mers (1,2-dihydroxybenzene, CT; 1,4-dihydroxybenzene, HQ, and 1,3-
dihydroxybenzene, RS) in water samples from the Czerniejówka River.
The device produced presented a robust linear response and sensitivity
of 0.0954 μA/μM (CT), 0.1241 μA/μM (HQ), and 0.6169 μA/μM (RS).
2.5. 3D printing
3D printing is an emerging technology with a remarkable capacity to
fabricate different geometries [36]. This technology is used in various
fields such as automotive manufacturing, aerospace, and medicine be-
cause of its high efficiency and precision. With this technology, complex
structures can be constructed at a low cost, and the design can be easily
changed, which is difficult to process by traditional techniques. Also, 3D
printers are commercialized at low cost, and some materials are also
cheap, which is attractive nowadays.
As a result of its flexibility, this technique has high potential in the
immobilization of enzymes. Nevertheless, enzymes immobilization
after the material is printed by adsorption or covalent bonding is a
method that has been proved before, in situ enzyme immobilization
(bio-printed, Fig. 5) is a novel technique recently used [14]. Liu et al.
[14] immobilized laccase by 3D bio-printed hydrogel with sodium algi-
nate (SA), acrylamide (AM), and hydroxyapatite (HA), and using agents
for covalent and crosslinking. They found that a lower ratio of AM re-
sults in not healthy gel formation, yet the addition of a high amount of
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International Journal of Biological Macromolecules 181 (2021) 683–696
AM collapses the surface network. HA showed to improve the substrate
conversion for this component enhance the permeability of the matrix.
The optimal ratio AM:HA:SA was determined to be 4:1.2:1. With these
conditions, 100% of the enzyme was retained, and no protein leakage
was observed. The laccase's stability was for 80% after 72 h of storage
and could be reused for 7 cycles for the remotion of p-chlorophenol
from water solutions. This technology could be improved by the addi-
tion of protection agents, which could increase enzyme stability.
2.6. Laser printing
Printing enzymes with inkjet have also been reported. For in-
stance, flexo printing enables large-scale manufacturing, using a
more comprehensive range of inks, and the printing can be used in
different materials. Flexographic ink consists of solvent, colorant,
and binder. The ink must have fast drying to make the process faster.
Paper is the most common surface. It is biocompatible, of low cost,
widely available, biodegradable, and has a porous structure. Authen-
ticity or oxygen indicator in food package indicator are possible ap-
plications for laccase immobilized on paper. Hurley et al. [37]
coated laccase and printed it on a paper from eucalyptus pulp; the
flexographic ink was a sulfo polyester resin dispersion HZ1100D.
Laccases were stable for 8 weeks and have 70% higher activity on eu-
calyptus pulp than on copy paper due to the material's porosity. After
printing on the paper surface, encapsulation of laccases in poly
(ethyeneimine) was evaluated using three methods rod coating,
screen, and flexo printing. In this case, the highest number of micro-
capsules was applied with screen printing technology due to the
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al. International Journal of Biological Macromolecules 181 (2021) 683–696

Fig. 4. Immobilization of laccase by MAPLE technique.

Fig. 5. In-situ Immobilization of laccase by 3D printing technique.

688
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
mesh opening is 450 μm, and with the other techniques is lower
(100–300 μm). The paper's surface was rinsed with water, and
most of the enzyme remained on the paper. The encapsulated en-
zymes preserved 63% of the initial activity after four weeks. Micro-
capsules preserve moisture and prevent unfavorable interaction
with ink components [38].
2.7. Metal-organic frameworks
Metal-organic frameworks (MOFs) are porous crystalline hybrid
materials conformed of metal ions or cluster and organic linkers.
These materials share characteristics of others, as mesoporous silica
and calcium carbonate. However, MOFs possess more thermal and
chemical protection for macromolecules inserted. MOFs have been
used in drug removal, dyes, and enzyme immobilization. Because
of their conformation, high surface area, and tunable pore volumes,
the transport of substrates through the enzyme is facilitated [39].
Zeolitic imidazolate frameworks (ZIFs) is a container that has been
studied, due to it has thermal and chemical stability, and does not
show cytotoxicity, this allows the preservation of enzymatic activ-
ity [40]. This process can be done by a post-synthesis or in situ im-
mobilization. Fig. 6 illustrate the preparation of MOFs by in situ
methodology.
Gascón et al. [41] developed a methodology to obtain enzyme-MOF
in one step employing an in situ approach at room temperature and
water as a solvent. This approach is relevant because immobilization is
given in one step, it is rapid, and its immobilization is high. By this
method, 100% of laccase was immobilized, preserving the activity of
the free enzyme. Likewise, with the use of MOFs, laccase activity could
be improved. Zhang et al. [42] studied the influence of cofactor Cu2+,
in the immobilization of laccase into MOFs (HKUST-1), and the activity
of laccase improved 150%, compared with free laccase.
Nevertheless, due to the enzyme-MOF composite possess a nano-
meter size, high dispersity, and low density, it is difficult to recover
them after the reaction process ends, and the possibility of recycling
is low [43]. To facile the separation of magnetic nanoparticles with
enzyme and MOF, Wu et al. [39] studied the effect on enzyme activity
in the immobilization incorporating functional groups (amino-
functionalized magnetic) in MOFs. They used a post immobilization
method (adsorption and covalent binding) found that 93.8% of the
laccase was immobilized.
Fig. 6. Preparation of MOFs by in-situ methodology.
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International Journal of Biological Macromolecules 181 (2021) 683–696
2.8. Crosslinked enzyme aggregates
Loss of activity due to enzyme's immobilization on a carrier has been
observed, especially with a high load of an enzyme. Crosslinked enzyme
aggregates (CLEAs) are an option for carrier-free immobilization. CLEAs
offer advantages like simple preparation, low-cost, easy to recover and
reuse method (Fig. 7). The preparation of CLEAs only needs precipitation
of the enzyme extract using precipitating agents, for this reason, this
method is considered cheap since a highly pure enzyme and a carrier are
not required [44]. However, one of the principal disadvantages perceived
is difficult of controlling the particle size and the handling [45]. Further-
more, the CLEA process's most critical part is to choose the correct
crosslinking agent and establish the adequate concentration, time, and
temperature. The combination of these parameters results in the CLEA's
particle size obtained. Another important consideration to take into ac-
count using this technique is as follows: i) low crosslinking agent concen-
tration will not allow the CLEA formation, ii) high crosslinking agent
concentration can impossibility laccase's flexibility affecting its activity
[45,46].
Normally, glutaraldehyde has been used as a crosslinking agent be-
cause it reacts with the enzyme's surface amino acids without modifica-
tion of the native structure [47–49]. Vršanská et al. [48] found that
CLEAs retained 80% and 74% of initial laccase activity isolated from
Trametes versicolor and Fome fomentarius, respectively. Also, good stor-
age and reusability were observed, up to 70 days in storage, keeping
the initial activity and ten cycles of use. Improvement of mechanical
properties and recycling of immobilized enzymes have been studied
to prepare magnetic CLEAs. CLEA is attached to amino-functionalized
magnetic nanoparticles and [45]. Primožič et al. [44] compared CLEA
and magnetic crosslinked enzyme aggregates (mCLEA). The immobili-
zation efficiency was 98.29% for CLEA laccase and 47.54% for mCLEA
laccase. Lower immobilization in mCLEA occurred as a response to an
incorrect stirring due to the magnetic particles. In both cases, after expo-
sure to supercritical carbon dioxide (SC CO2) the residual activity was
improved.
2.9. Hybrid nanoflowers
Hybrid nanoflowers (hNFs), are structures that mix an organic com-
ponent (enzyme) with an inorganic one (metal). Ge et al. [50] observed
the formation of a porous and flower-like structure in the synthesis of
protein-inorganic nanostructures, with the presence of Cu+2. The
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
Fig. 7. Preparation of CLEAs. (a) General crosslinking method, (b) polymer based crosslinking method and (c) magnetic CLEAs.
growth of nanoflowers occurs as a response to the interaction between
amino acids and copper ions. Suppose the enzyme is used instead of the
protein component, the enzyme activity and stability increase [51]. En-
zymes present a strong affinity to metal ions as a cofactor. The formation
is completed through three steps: nucleation, growth, and completion
(Fig. 8). This structure provided enhanced activity, highly stable at dif-
ferent pH, temperatures, cycles of use, and storage periods [52]. An in-
crease of 650% of the activity of laccases immobilized in hNFs in
contrast with bulk or free enzyme has been reported. The principal ad-
vantages of hNFs immobilization are a better mass-transfer between the
enzyme and the substrate due to the extensive surface area generated,
favorable enzyme conformation, sensitivity, and selectivity [52].
Recently, it was found that the incorporation of amino acid in the
synthesis of nanoflower help to stabilize the conformational structure
of the enzyme. Maurya et al. [53] synthesized a hybrid nanoflower by
mixing an aqueous CuSO4 solution with phosphate buffer containing
laccase and lysine. Due to conformational changes in the structure of
laccase after conjugation with lysine, the activity was increased to
270%, with 71% immobilization yield, and it was able to decolorize 89%
of Reactive Blue 4 in 1 h. Although all the advantages that this system
presents, the reuse of hNFs is still a problem. For this reason, their appli-
cation is not widely extended yet [54]. Han et al. [55] include Fe3O4
magnetic nanoparticles in the synthesis process of hNFs with
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International Journal of Biological Macromolecules 181 (2021) 683–696
horseradish peroxidase, to facilitate their reuse. In this study, the encap-
sulation efficiency was 84.16%, with an enzyme activity of 183%. Some
authors had reported a lower affinity of the substrate when the en-
zymes are immobilized in magnetic supports. Fu et al. [54] also prepared
magnetic nanoflowers, using laccases as an organic component. The
laccase loading activity in magnetic hNFs was about 270% compared
to free laccases, whereas the laccase in hNFs was 330%. The difference
between the two systems might be that the magnetic nanoparticles
could cover some laccases' activity sites. In both cases, after the use,
the enzymes were quickly recovered by an external magnetic field.
3. Novel carriers for immobilization
Enzyme immobilization using a carrier or support is the most com-
mon form of immobilization. In this case, the immobilization could be
by adsorption or by covalent bonding. Different materials are available
as enzymatic support, and the proper support's election determines the
biocatalytic efficiency. The support material must have a highly porous
structure. The material must also have an affinity for the enzymes or be
first modified to form functional groups to increase the enzyme affinity.
Nowadays, research has been performed in thermostable materials, resis-
tant to contamination, biodegradable, and available at low cost [56].
Table 2 summarizes various techniques for laccase immobilization.
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al. International Journal of Biological Macromolecules 181 (2021) 683–696

Fig. 8. Schematic development of hNFs.

3.1. Biomaterials
Different materials, especially “biomaterials” (agarose, starch, chito-
san, cellulose, alginate), have been used to immobilize different enzymes.
These materials have been studied because they are environmentally
friendly, easy to modify, cheap, easy to produce, high surface area, and
have a wide variety of functional groups on their surface [8,17,57–61].
Agarose has been proven to be a promising material to immobilize en-
zymes, it is considered inert support and has biocompatibility [12,62].
Monoaminoethyl-N-aminoethyl (MANAE-agarose) gel proved to en-
hance the properties of laccases due to its primary amino groups, which
are very appropriate for immobilization, Brugnari et al. [63] immobilized
laccases in this gel, the activity retention value was 138%. The
immobilized enzyme retained >90% of its initial capability to degrade

Table 2
Techniques for laccase immobilization.

Strain Technique Type immobilization Retain Storage stability Reuse cycles References
activity
(%)

Aspergillus oryzae 3D printing Crosslinking and covalent 100 33% 7 [14]

Trametes Versicolor ESI Entrapment/adsorption 100 90 days 100% activity room temperature 25 [19]
Trametes versicolor Electrospinning Covalent 71 7 days 4 °C 7 [25]
Trametes versicolor Electrospinning Encapsulation 100 65.8% after 30 days [30]
hNFs Crosslinking 200 30 cycles [82]
Rhus vemicifera hNFs Crosslinking 165 4 °C 80% 30 days 10 cycles 85% [83]
Trametes Versicolor magnetic hNFs Crosslinking 270 4 °C 92% after 60 days 5 cycles 95% [54]
Trametes Versicolor hNFs Crosslinking 270 24 days, 61% 6 cycles [53]
MOFs Adsorption and covalent binding 83.3 After 28 days 89% 3 cycles [39]
Trametes versicolor MOFs Encapsulation 154 4 °C 70% after 30 d After 10 cycles 78.9% [42]
Aspergillus oryzae MOFs Encapsulation 98 [41]
Trametes versicolor MAPLE Adsorption 4 °C, 100 days 5 [33]
Cerrena unicolor C-139 SPP Cross-linking, adsorption 59 8 days 44% of activity 7 [21]
Trametes versicolor Laser printing Adsorption 4 °C 90% after 35 days [84]
Trametes versicolor Flexographic Adsorption 100% 8 weeks [37]
Printing
Trametes versicolora CLEAs Crosslinking 80% after 70 days 12 cycles [48]
Fomes Fomentariusa 74%
Cerrena unicolor Magnetic CLEA Crosslinking 46.8% [49]
Trametes versicolor CLEA Crosslinking 98.29% [44]
Magnetic CLEA 47.54%
a
Laccases from native isolates fungi.

691
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
BPA after 15 cycles, demonstrating its efficiency in the biotransformation
process.
Chitosan, a natural biopolymer, is acquiring acceptance as a support
because of its availability, low cost, easy modification. It is biodegrad-
able because it is typically obtained from chitin, which exists in the
cell fungi and skeleton. Also, chitosan has efficient structural properties
as high mechanical strength, good affinity to proteins because it pos-
sesses reactive groups -NH2 on the surface that can be used for enzyme
immobilization [29,64]. However, to increase the immobilization
crosslinking agents are used as glutaraldehyde. For example, Bilal et al.
[62] immobilized laccase on chitosan beads by covalent bonding with
glutaraldehyde, obtaining a maximum immobilization efficiency of
84.7%. In this material, the residual activity was 89.31% after five reus-
ability cycles.
Nevertheless, it also has been observed that glutaraldehyde could af-
fect the native conformation of enzymes, causing a decrease in the activ-
ity or even the deactivation.
For this reason, the incorporation of inorganic materials as clays and
active carbon can improve enzyme immobilization. Mehandia et al. [65]
immobilized laccases on two systems, one was on crosslinked chitosan
beads (CB) using glutaraldehyde and chitosan-clay composite beads
(CCB). The yield of immobilization was higher in the CCB (88.4%) and
the CB (83.6%), possibly because the laccase has better compatibility
Fig. 9. Agro-industrial waste for laccase immobilization.
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International Journal of Biological Macromolecules 181 (2021) 683–696
with the chitosan and the clay. Other parameters as surface area,
pore-volume, density, thermal stability, and reusability were increased
in the CCB.
Starch is also considered to be proper support due to it was easy to
functionalize. Chen et al. [66] functionalized maize starch with
glucoamylase and α-amylase, conferring high surface areas
representing a high potential for eliminating pollutants. The immobili-
zation was made by adsorption and crosslinking with a yield of 64.8%.
Cellulose is another biomaterial that has been studied for enzyme im-
mobilization. It is considered a cheap material extracted from nature
and abundant polysaccharide sources. It has many hydroxyl groups on
the surface that facility bonding immobilization. Cellulose nanocrystals
(CNCs) have been used as an adsorption method to eliminate dyes.
However, it is needed to enhance their adsorption capacity, and
polydopamine, a surface modification method, was studied. The modifi-
cation increases the interaction with dyes facilizing their adsorption.
Wang et al. [67] prepared CNCs with PDA. High adsorption capacity per-
mitted the removal of 500 mg/L of methylene blue at 5 min, and CNCs
could be regenerated to maintain their adsorption capacity for four cy-
cles. Bacterial cellulose (BC) is cellulose synthesized by some bacteria
as Gluconacetobacter xylinus. It is a highly porous material that has
been used to entrapment laccases and has demonstrated excellent per-
formance as bio-colorant support, with immobilization of 88% [68,69].
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al.
3.2. Agro-industrial waste materials
For immobilization, using synthetic support materials can be an ex-
pensive method. Also, traditional supports are not biodegradable or en-
vironmentally friendly, representing a problem for their disposition
when the process ends (Fig. 9). For these reasons, more investigations
are focused on developing inexpensive, biodegradable, and easy-to-
finding materials that can be functionalized and used for the immobili-
zation of enzymes. One alternative is agro-industrial wastes (Fig. 10),
which are defined as the residues generated from the growing and pro-
cessing of agricultural products and household waste, representing an
increasing concern [17,70]. Different materials that have been used for
laccase immobilization are summarized in Table 3. For example, chicken
feathers represent a problem on their final disposition. Due to their
characteristics, new applications have been developed; one of them is
in enzyme immobilization. Immobilization of laccases into chicken
feathers via covalent attachment was studied by Suman et al. [71].
Chicken feathers were firstly functionalized with (3-aminopropyl)
trimethoxysilane (APTMS), and glutaraldehyde was used as a
crosslinker to bond laccases. After, laccases were put in contact, and
74.24% of immobilization yield was obtained. The reusability aspect of
the immobilized laccases on chicken feathers was studied using 2,2′-
azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate,
and until eight consecutive cycles, the activity was maintained at 95%
[71].
Eggshell is a biocompatible, safe, and cheap by-product with high
porosity, large surface area, and due to its compositions containing dif-
ferent organic groups that give the possibility to be an ideal support for
enzymes. Girelli et al. [72] used eggshell for immobilization of laccase by
two protocols immersion and dropping immobilization. The enzyme-
dropping protocol by crosslinking a residual activity of 45% was
achieved and used for 6 cycles. Coconut fiber is an abundant product
in many regions of the world. It has physical-chemical properties of
great potential in different applications such as biotechnological pro-
cesses, particularly potential support for enzyme immobilization [70].
Two methods of immobilization have been proved with laccases. In
the adsorption immobilization method, the laccases were directly put
in contact with the coconut fiber. Around 45% of immobilization yield
was obtained, and low enzyme concentration was possible to
immobilized, due to coconut fiber does not have a porous structure,
and it has a low surface area, this limit the molecules to be immobilized
Fig. 10. Agro-industrial wastes used for laccase immobilization and their advantages.
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International Journal of Biological Macromolecules 181 (2021) 683–696
on the surface [73]. Bezerra et al. [74] immobilized laccases on coconut
for clarification in juice in covalent and physical immobilization. The ac-
tivation of coconut fiber was with ethylenediamine and the bonding
with glutaraldehyde. Laccases were chemically modified with EDA.
98% of laccase was immobilized, the laccases immobilized reduced
61% of turbidity and 21% of original color, retaining 100% of the initial
activity after 10 cycles.
Cellulose nanofibers (CNFs) extracted from sugar mill bagasse, an
abundant available agricultural waste, were used for immobilized
laccase. CNFs were functionalized by a glutaraldehyde treatment
obtaining an 85% immobilization. 100% of degradation of trifluralin
was obtained in 24 h [75]. Spent grain is the primary waste from the
beer industry. It has a high nutritive value, rich in protein and fiber.
The surface of this grain consists of numerous fibrous tissues. It contains
numerous functional groups as carboxyl, hydroxyl, and amino, and it is a
low-cost product. It was suggested to be a potential carrier for enzyme
immobilization [70]. Two methods immobilized a commercial laccase,
adsorption (on spent grain and digested spent grain) and covalent
bonding (using glycidol or glycidol with ethylenediamine). The best-
recovered activities were for the immobilization on digested spent
grain (90%), having until 10 cycles of continuous operation [76]. To facil-
itate their recovery, magnetic properties have been added to the spent
grain [77].
Biochar is a carbonaceous material obtained from the pyrolysis of
biomass. Low cost, porous structure, high surface area, high water sta-
bility are advantages of this material [78,79]. Naghdi et al. [80] used bio-
char as a carrier for immobilization of laccases. Different acid treatments
for nanobiochar were proved. The best combination was with H2SO4/
HNO3. After this treatment, more concentration of COOH functional
groups was available. However, laccase was not high binding (26%).
Micro-biochar (biochar with particle size < 100 nm) offers a high sur-
face area than biochar. Lonappan et al. [79] used functionalized micro-
biochars obtained from pine wood, pig manure, and almond shell
using glutaraldehyde for covalent bonding. A functionalization with an
organic acid (citric acid) was made before the immobilization process
for introducing -COOH functional groups on the supports. In the three
materials was observed an immobilization. However, the material
with more immobilization was pig manure micro-biochar, 34.1 U/g,
due to this material possess a larger surface area than the others. The
ability of removal with these immobilized systems and the reuse was
proved with diclofenac. Almost 100% of removal was obtained with
L. Alvarado-Ramírez, M. Rostro-Alanis, J. Rodríguez-Rodríguez et al. International Journal of Biological Macromolecules 181 (2021) 683–696

Table 3
Materials used as support for laccase immobilization.

Material Application Stability References

Green coconut fiber Clarification of apple juice 100% activity after 10 cycles [74]
61 ± 1% color
Turbidity 29 ± 1%
Pine wood (BC-PW) Removal of diclofenac 40% activity after 5 cycles [79]
Pig manure (BC-PM) 500 μg/L in 2 h
Almond shell (BC-AS)
Biochar Removal of carbamazepine 11% after 7 cycles [80]
(produced by pyrolysis of biomass) 83% 24 h
20 ng/mL
Chicken Feather Oxidation of Veratryl Alcohol 2 mM 100% 48 h 94.32% after 3 weeks of storage [71]
Spent grain ABTS 10 cycles [76]
Alginate Laccases packed bed bioreactor for decolorization 100% until 5 cycles [85]
continuous effluent water Retains its efficiency even after 120 h of continuous
use.
Sodiumalginate (SA) and acrylamide (AM) Removal of p-chlorophenol 80% of its initial activity during 72 h. Was able to be [14]
hydroxyapatite (HA) 32.1% reused for 7 batches
Carbon black-nanomodified Bio-sensor catechol 25 measurements on the same electrode, 3 months [19]
at room temperature
MANAE–agarose Degradation BPA >90% of initial capacity to degrade BPA after 15 [63]
100% cycles
Chitosan/poly (vinyl alcohol) fibers Time-temperature indicator After 10 days retained 94.53% [29]
Chitosan Decoloration of five dyes 80.19% after 10 cycles [64]
Hippospongia communis spongin-based Biodegradation of bisphenol A (BPA) 100% bisphenol F 80% of its initial activity after 50 days of storage [81]
scaffold (BPF) bisphenol S (BPS) 40%
Maize starch Pesticides removal atrazine and prometryn 61% 7 days [66]
Eggshell Syringic acid degradation 6 cycles 30% [72]
Cellulose Degradation of trifluralin [75]
Cellulose Removal of methylene blue 4 cycles [67]
Bacterial cellulose Coloration of flax fabrics 3 cycles of washing [68]
Bacterial cellulose ABTS 7 cycles [86]

the three systems in less than 6 h, and retained activity was 40% after 5
cycles.
Ramírez-Montoya et al. [56] prepared carbon supports from ligno-
cellulosic wastes (pecan nutshells, peach stone, pistachio shell, and
pine nutshell) that generally are disposed of in dumps. Carbon prepared
was used for immobilized laccases from T. versicolor, and it was carried
out by adsorption. The immobilization capacity of the material
depended on the mesopore volume. Pecan nutshell was the most effi-
cient support having an adsorbing capacity of 10 mg of protein per g
of support. This support was proved for decolorization of AO7 70% of de-
colorization was obtained. However, the principal mechanism involved
was adsorption. Hippospongia communis spongin-based scaffold was
proved to be compatible with enzymes and no required additional sur-
face functionalization. Also, the morphologic proved to be a good struc-
ture where the access to active sites of enzyme are easier to access, and
the groups in the structure of the material (carboxyl, carbonyl and hy-
droxyl) not only improves the attachment to the surface, also increased
the transfer of electrons, making the degradation of contaminants more
effective. Zdarta et al. [81] proved the capacity of biodegradation of
bisphenol A (BPA) and Bisphenol S (BPS) with laccase immobilized on
this material, obtaining 100% and 40% respectively, and the reusability
was over 40% after five cycles.
4. Concluding remarks
New technologies have been developed to increase the functionality
and structural availability of laccases in several fields. For instance, some
use enzymes as biosensors, bioanalytic tools, and industrial biocatalysts.
Nowadays, immobilized enzymes have been used to reduce costs in the
biotechnology industry. There are a wide variety of methods that can be
used to achieve highly effective immobilized enzymes. Nevertheless,
some of them have more advantages, and others need to improve
their operating conditions. The material presented in this review
shows brand new technologies used to immobilize enzymes and seeks
to be a helpful guide to the scientific community when choosing en-
zyme immobilization techniques. It is essential to combine techniques
to allow immobilization process quickly, using a small amount of en-
zyme with improving efficiency, and in some cases, using biodegradable
and low-cost materials.
Nowadays, there is an increasing tendency to studies focused on bio-
degradable materials that do not harm the environment. Moreover, due
to the circular economy representing a significant role in the global
agenda, it is essential to use agro-industrial wastes. In addition, the ex-
ploitation of agro-industrial wastes as enzymes supports could be useful
in maintaining the overall cost-effective ratio of the entire process. Ap-
propriate deployment of nanotechnologically engineered matrices for
enzyme immobilization can open the possibility of using these systems
at an industrial scale.
Declaration of competing interest
Authors declare no conflicting interests.
Acknowledgments
The PhD scholarship awarded by Consejo Nacional de Ciencia y
Tecnología (CONACYT), Mexico to Lynette Alvarado Ramírez (486638)
is thankfully acknowledged. We are also thankful to “Programa
Iberoamericano de Ciencia y Tecnología para el Desarrollo” in the Latin
American development network “Lacasas Inmovilizadas para la
Degradación de Compuestos Aromáticos en Aguas Residuales” (LIDA,
project 318RT0552) and to i-Link+ program funded by CSIC-
Tecnologico de Monterrey (“Contaminantes emergentes y prioritarios
en las aguas reutilizadas en agricultura: riesgos y efectos en suelos,
producción agrícola y entorno ambiental” LINKB20030). All listed au-
thors are also grateful to their representative universities for providing
literature access. Figs. 1–10 were created with BioRender.com
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