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Enzyme-loaded nanoparticles for the degradation of wastewater

contaminants: a review

Article  in  Environmental Chemistry Letters · January 2021

DOI: 10.1007/s10311-020-01158-8

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Environmental Chemistry Letters
https://doi.org/10.1007/s10311-020-01158-8

REVIEW

Enzyme‑loaded nanoparticles for the degradation of wastewater

contaminants: a review
V. Karthik1 · P. Senthil Kumar2   · Dai‑Viet N. Vo3 · P. Selvakumar4 · M. Gokulakrishnan1 · P. Keerthana1 ·
V. Audilakshmi5 · J. Jeyanthi6

Received: 26 November 2020 / Accepted: 14 December 2020

© The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature 2021

Abstract
Preventing water pollution and conserving water are major issues in the context of population growth and worldwide pol-
lution, calling for advanced remediation techniques. Classical remediation techniques of water cleaning such as membrane
adsorption are able to separate pollutants from water, yet the separated pollutants require additional treatment or disposal.
Therefore, techniques that degrade the pollutant appear promising, provided that pollutants are organic and degradable. Here,
we review the enzymatic degradation of organic pollutants with focus on methods to immobilize enzymes and nanoparticles
as support materials. We discuss the degradation of pesticides, dyes, phenolic compounds and antibiotics.

Keywords  Enzyme · Nanoparticles · Toxic pollutants · Wastewater · Immobilization · Removal

Abbreviations CV Crystal violet

2,4-DCP 2,4-Dichlorophenol DCP Dichlorphen
AB92 Acid blue 92 DG6 Direct Green 6
ABTS 2,2′-Azinobis (3-ethylbenzothiazoline- DR31 Direct Red31
6-sulfonic acid) DWCNTs Double walled carbon nanotubes
BG Brilliant green EDC 1-Ethyl-3-(3-dimethyl aminopropyl)
BPA Bisphenol A carbodiimide
CLEA Cross-linking enzyme aggregate ENPs Enzyme nanoparticles
CLEC Cross-linking enzyme cluster Fe3O4 Iron oxide
CNTs Carbon nanotubes GO Graphene oxide
CP Chlorophene GpdQ Glycerophosphodiesterase
CTAB Cetyltrimethylammonium bromide GRM117 Ganoderma lucidum
H2SO4 Sulfuric acid
HCL Hydrochloric acid
* P. Senthil Kumar
senthilchem8582@gmail.com; senthilkumarp@ssn.edu.in His6-EcPepQ His6-tagged Escherichia coli prolidase
HRP Horseradish peroxidase
1
Department of Industrial Biotechnology, Government ILTNs Immobilized laccase in Titania
College of Technology, Coimbatore 641013, India nanoparticles
2
Department of Chemical Engineering, Sri Sivasubramaniya K2S2O8 Potassium persulfate
Nadar College of Engineering, Chennai 603 110, India KMnO4 Potassium permanganate
3
Center of Excellence for Green Energy and Environmental LiP Lignin peroxidase
Nanomaterials (CE@GrEEN), Nguyen Tat Thanh University, MCLEAs Magnetic cross-linked enzyme aggregates
Ho Chi Minh City, Vietnam
MG Malachite green
4
Department of Chemical Engineering, Adama Science MGO Magnetic graphene oxide
and Technology University, 1888 Adama, Ethiopia
MNPs Magnetic nanoparticles
5
Department of Biotechnology, KIT-Kalaignar Karunanidhi MWCNTs Multi-walled carbon nanotubes
Institute of Technology, Coimbatore 641402, India
NHS N-hydroxy sulfosuccinimide
6
Department of Civil Engineering, Government College NPs Nanoparticles
of Technology, Coimbatore 641013, India

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 Environmental Chemistry Letters

OP Organophosphorus several disadvantages like less stability, and inability to be

P2O5 Phosphorous pentoxide recycled from the reaction mixture. The enzyme molecules
PLO9 Pleurotusostreatus are confined onto or within a support or matrix physically
SDAs Structure directing agents or chemically or both, as a means of immobilization. Hence,
Si MNPs Silica-coated magnetic nanoparticles most of its activity can be restored. Different techniques used
SiO2 Silica oxide for immobilization include microencapsulation, covalent,
SWCNTs Single-walled carbon nanotubes adsorption, attachment, and entrapment (Hu et al. 2015).
TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl Immobilization can be done on various materials, which
TEOS Tetraethyl orthosilicate are called carriers or support materials. Not all materials
TiO2 Titania can be used as carriers but carriers do have certain ideal
characteristics such as insolubility under reaction conditions,
high affinity to enzymes, chemical stability, thermal stabil-
Introduction ity, regeneration, reusability, and biocompatibility (Sardar
2015). One such carrier with all such properties is nano-
Billions of gallons of wastewater are produced and released materials. In recent days, nanoparticles gain more attention
every day, which in turn becomes a threat to nature. Moreo- than the existing various kinds of supports due to their supe-
ver, the limited possibility for the supply of freshwater to rior properties of lower resistance to mass transfer, higher
meet the demands of increasing population throughout the catalytic stability, and higher storage stability (Pinto et al.
world has urged us to conserve water in all possible ways, 2015). In addition, nanoparticles have high enzyme density
including the efficient and advanced strategies of wastewater and surface area (SA)-to-volume ratio (Kim et al. 2016). A
treatment. By treating the wastewater, nature is restored with wide variety of nanosupports, including magnetic and non-
desirable water quality. Many chemical/physical wastewater magnetic nanomaterials, can be used for the immobilization
treatment systems are used to treat wastewater before it is of enzymes to remove toxic pollutants from wastewater.
discharged (Surendhiran et al. 2017). However, they remain
ineffective in providing adequate safe water. Recently, the
technological leap in enzyme-based treatment has purified
the water to the extent of portability, by operating under Nanomaterials
mild environments.
Enzyme engineering has developed to an extent where Properties
we are readily evolving with new techniques to find effi-
cient solutions for most of the complications in each field. Nanoparticles are microscopic particles with at least one
Enzymes being a biocatalyst, speed up many chemical and dimension (height, length or breadth) of the particle of less
biochemical reactions and have vast applications in the phar- than 100 nm, mostly in the range of 1–100 nm. Because of
maceutical industry (Apetrei et al. 2013), diagnosis, textile the nanoscale size, nanomaterials possess some unique phys-
industry (Srivastava et al. 2020), biofuel industry (Selva- ical and chemical properties (Gatoo et al. 2014) as follows:
kumar and Sivashanmugam 2018a, b, 2019), food industry
(Ismail and Nielsen 2010), etc. Additionally, immobilized 1. Smaller size.
enzymes have the most advantages over free enzymes. 2. High surface-to-volume ratio.
Immobilized enzymes are made stable through various 3. High solubility.
processes, which are further systematically discussed in 4. High mobility in solution.
this review article. These advantages include immobilized 5. High strength.
enzymes’ capability to be reused after recovering from the 6. High conductivity.
reaction mixture, retention of enzyme activity for a more 7. In addition, nanoparticles show high mobility in solu-
extended period. On the other hand, free enzymes are com- tion, enhanced redox, and photocatalytic properties.
paratively unstable, and there are certain cons when it comes
to industrial applications. This article has discussed the These properties made nanoparticles to be used in many
manipulation of enzymes (immobilization) in wastewater fields like environmental sciences for the detection (Forouhi
management (Tonini and Astrup 2012), particularly solid et al. 2017; Sithara et al. 2017), monitoring and removal
wastes treatment and wastewater purification. The removal of pollutants (Joshiba et al. 2020; Datta et al. 2020), in life
of toxic compounds from water is a very much needed tech- sciences for drug delivery (Karimi 2018; Selvakumar et al.
nology in this contemporary world as the pollutants are 2018), diagnostics (Mukherjee 2019), nutraceuticals, and the
increasing with the increase in population and urbaniza- production of biomaterials (Moyo et al. 2013).
tion. It is widely known that the free enzymes also have

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Nanomaterials as immobilization carriers attributes are enzyme density (Pavlidis et al. 2012), mobility
(Algar et al. 2012), and nanoparticle morphology (Campbell
The characteristics of the immobilization matrix play a vital et al. 2014). The advantages and disadvantages of the usage
role. The general characteristics of immobilized matrix are of nanoparticles are discussed in Table 1.
presented in Fig. 1. The ideal matrix should possess physical
resistance to compression, inertness toward enzymes, hydro- Enzyme density
philicity, biocompatibility, and high strength. In addition, it
should be readily available at a low cost. During enzyme immobilization on nanoparticles, the activ-
Natural polymers like cellulose, chitin, alginate, and ity of the enzyme increases with an increase in enzyme den-
collagen are widely used (Rodríguez-Restrepo and Orrego sity or the number of enzymes on the surface of the nano-
2020). For example, immobilization of dextranase using particles up to an optimum level, after which the increase
alginate increases reusability up to 7 cycles after performing in enzyme density decreases the activity; this is because of
catalytic activity (Shahid et al. 2019). Synthetic polymers interaction with the adjacent enzyme molecules and con-
like silica and zeolites are also used for various applications. formational deactivation (Bosley and Peilow 2000). This
For example, immobilization of enzymes on porous silica can be controlled by the functionalization of the surface of
as a biosensor (Hartmann and Kostrov 2013). Even though nanoparticles (Pavlidis et al. 2012).
many natural and synthetic polymers are available, carriers
with high surface area-to-volume ratios are preferred. Nano- Mobility
particles have high surface area-to-volume ratios and are
efficient carriers. Although immobilizing enzymes in nano- When enzyme-conjugated nanoparticles are dispersed in
particles are complex, some characteristics of the immobi- the solution, the nanoparticles show Brownian motion. This
lization matrix could enhance enzyme performance. These motion is responsible for the high activity compared to free

Fig. 1  Characteristics of the immobilized matrix play a vital role in enzyme immobilization. Hence, the immobilized matrix selection is mainly
based on physical and chemical properties and other commercial considerations such as cost and availability

Table 1  Advantages and disadvantages of using nanoparticles as immobilization carriers

Advantages Disadvantages

Higher surface-to-volume ratio Large-scale implementation

Higher mechanical strength (Gatoo et al. 2014) Recovery of nanoparticles after usage
Higher mobility (Brownian motion) (Algar et al. 2012) Cost of fabrication
Effective enzyme loading
Less mass transfer resistance
Able to maintain enzyme orientation and enzyme density through surface chemistry (Pavlidis et al.
2012)

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enzymes. The weak interaction between nanoparticles and the total production cost of enzyme-mediated reactions. As
substrate leads to an increase in the substrate concentra- previously discussed in “Properties” section, immobilization
tion around the nanoparticles, ultimately enhancing activity requires carrier or support material and its characteristics
(Algar et al. 2012). influence the rate of reaction and immobilization.

Nanoparticles morphology Modes of immobilization

Nanoparticle morphology plays an important role in enhanc- Immobilization can be done in different methods and rep-
ing enzymatic activity. Nanoparticles show a high surface resented in Fig. 2. The variation in these methods depends
area due to their smaller size. This allowed the material on the physical retention method and the chemical bonding
to have a large center-to-center distance with the adjacent method. As the physical bonding is comparatively weak,
immobilized enzyme moiety. This reduces protein-to-pro- it will sustain the substrate binds for a shorter period and
tein interaction and inactivation of enzymes (Campbell et al. hence the preference for chemical bonding is high. The phys-
2014). ical methods are characterized by weak interaction forces
(Jegannathan et al. 2008). Chemical bonding gives internal
stability, which can last longer (Gao and Kyratzis 2008). The
Enzyme immobilization immobilization mode also depends on the carrier material
chosen. In our article, the nanoparticles are the carriers, and
As free enzymes cannot be easily extracted or isolated from the possible methods of immobilization using nanoparticles
the product and are lost after the first use, free enzymes’ as carriers are discussed below.
usage is avoided in many industries. The immobilization
of enzymes overcomes this disadvantage and thus leads to Physical entrapment
revolution. Enzyme immobilization is the technique in which
the enzymes are imprisoned or confined in distinct support In the physical entrapment method, there is no direct attach-
or matrix to restrict the freedom of enzyme’s movement. ment of the enzymes to the carrier surface; instead, each
This process increases stability as it limits enzyme’s ther- molecule is dispersed within the matrix. The diffusion of the
mal activity at higher temperatures, which also makes it less enzyme is constrained, whereas the substrate and product are
vulnerable to denaturation at higher temperatures. It also allowed for complete movement.
increases the resistance to changes in pH, and temperature Enzymes are homogeneously dispersed within the poly-
during industrial processes. Other advantages include the meric network (scaffold). Physical entrapment is an irre-
ease of separation of enzymes from the final product and the versible method and the enzymes are entrapped within the
separated enzymes are capable of being reused. This reduces support.. The enzyme will not interact chemically with the

Fig. 2  Enzyme immobilization
can be done using four methods:
adsorption, entrapment, cross-
linking, and covalent bonding

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Environmental Chemistry Letters

polymer. Alginate, collagen, silicon rubber, gelatin, carra- anchoring the chemical to the carrier and interacting physi-
geenan, polyurethane, polyacrylamide, and polyvinyl alcohol cally with the enzyme. Examples of such modifying agents
with styryl pyridinium group (Guisan 2006) can be used are bifunctional carbonyl compounds. The most common
as matrices for the entrapment method. To be more spe- modifying agent is glutaraldehyde (Rios et al. 2019).
cific, alginates are the most commonly used matrix because As described above, the enzyme-carrier affinity and sur-
of their properties, such as mild gelling and non-toxicity. face area of carriers play an essential role in adsorption. The
The entrapment of enzymes can be approached in various support material is kept in contact with the enzyme solution
methods such as gel entrapment, fiber entrapment, micro- for a particular or fixed time under favorable conditions so
encapsulation, electropolymerization, photopolymerization that they are capable of sustaining enzyme activity. By wash-
and sol–gel processes for the lattice. Because of the limita- ing with a buffer, enzyme molecules that are not absorbed
tions of mass transfer, practical applications are restricted. can be eliminated from the surface. This simplest method
The entrapment method can also be done in two consecu- is economical and involves reversible linkages between
tive steps as follows: the support material and the enzyme used. Leakage of the
enzyme is one of the major disadvantages of the adsorption
1. Mixing enzymes with monomer solutions. method. Desorption and inactivation are also an issue in
2. Polymerization of the solution either by chemical this technique.
method or changing the conditions of the experiment.

The optimal pH, polarity, or amphilicity are the ideal Covalent binding
microenvironment that could be maintained. This is done
using substances such as sol-gels, polymers, polymer/sol–gel Covalent binding is the technique in which the immobiliza-
composites, and other inorganic materials (Datta et al. 2013; tion of enzymes is carried out by forming chemical bonds
Tsai and Doong 2007). The entrapment increases mechani- between the non-essential chemical group of enzymes and
cal stability and enzyme leaching can be avoided to an the support material chemical group. Usually, in the bind-
extent (Shen et al. 2011). The denaturation of enzymes is ing of enzymes, the functional group is bonded utilizing the
also avoided. Besides, the physicochemical environment side chains of lysine (ϵ-amino group), cysteine (thiol group)
can be maintained between the immobilization material and glutamic and aspartic acids (carboxylic group) (Guisan
and the enzyme. Rather than its advantages, this method’s 2006; Soleimani et  al. 2012). The enzyme’s covalently
challenges include enzyme leakage in some cases when the bonded activity depends on the shape, size, and composition
support material consists of larger pores, which causes the of the support material, nature, and conditions maintained
deactivation of the immobilization. The obsession of the car- during coupling methods (Mohamad et al. 2015). The cou-
rier and low loading capacity is also the disadvantage of this pling reactions may be of the following different reactions.
method. The pore size factor is equal to the immobilized
particle size/pore size of the support material. This factor is 1. Isourea linkage formation.
essential to avoid enzyme leakage. 2. Diazo linkage formation.
3. Peptide bond formation.
Adsorption 4. An alkylation reaction.

Adsorption immobilization is the method in which the If the support material is insoluble, there is an advantage
enzyme is attached to the carrier surface by weak forces, that the binding of the enzyme to the support matrix would
i.e., physical interactions, namely van der Waals force, be irreversible. As vigorous reactions destroy the active con-
hydrophobic interaction, hydrogen bond, and an electrostatic formation of enzymes and carriers, the immobilization reac-
force (Jegannathan et al. 2008). The existence of specific tion is to be carried out in mild conditions only. The direc-
functional moieties on the surface of both the enzyme and tion of enzyme binding is an important factor in covalent
the carrier is a necessary prerequisite for successful adsorp- binding as it determines the stability. Covalent binding can
tion. These functional groups help in the strong binding of be done as a single step process when the functional group
the enzyme and carrier, restricting the chemical modifica- in the support material does not require any modification.
tions due to environmental changes or certain chemical reac- Otherwise, it requires two steps as follows:
tions. If these functional groups are absent, the carrier is then
subjected to chemical modification. This can be achieved 1. The functional groups present on the carrier material are
using the modifying agent. The characteristics of the mod- to be actuated by a specific reagent.
ifying agent include the presence of at least two reactive 2. Addition of enzyme for the coupling reaction (common
groups in its molecule. Each group has a specific function in step).

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 Environmental Chemistry Letters

Cyanogen bromide is the most commonly used reagent Hence, enzymes are precipitated from the solution to
for activation of the covalent binding process. Covalent form physically clustered protein molecules.
bonds form a powerful binding between the lipase and the
carrier matrix. It also makes it easier to reuse (Sheldon 2. Cross-linking enzyme crystal.
2007a, b; Ovsejevi et al. 2013) and avoids the release of
enzymes into the environment (Ispas et al. 2009) than in
other immobilization methods. In one study, functionalized This method is similar to that of cross-linking enzyme
­Fe3O4 nanoparticles modified using carboxymethylated aggregate (CLEA), except that it forms crystals. This leads
chitosan were progressed and used as carriers for papain’s to improved mechanical properties but does have disad-
covalent conjugation process (Liang and Zhang 2007). The vantages that can be overcome by the CLEA method. The
magnetically immobilized papain expressed better super- above reactions for the immobilization of enzymes can be
paramagnetism properties and enhanced the activity of the in various methods like mixing prepolymers with photosen-
enzyme. It also provided better tolerance to the differences sitizers, for example benzoin ethyl ether, then melting and
in medium pH and temperature and increased storage stabil- mixing it with an enzyme solution. This uses near-ultraviolet
ity and reusability. In this method, the confinement strength radiation for gelling or freezing of monomer solutions con-
is high and the leakage of enzymes is also restricted to an taining enzymes to form small beads. These processes are
extent. The thermal stability and half-life of enzymes are then accompanied by polymerization. The process is started
found to increase when they are coupled with various carri- by gamma radiation. The enzymes are mixed in a buffered
ers like mesoporous silica, chitosan, etc. (Ispas et al. 2009). aqueous solution containing the acrylamide monomer and
However, it also has its defective consequences when there a cross-linking agent before polymerization initiated by
is a multipoint (numerous link) attachment to the support chemicals is performed (Bashir et al. 2020). It is known that
and steric hindrance of enzymes. The support material appli- the CLEA method is the most preferable as it can be read-
cable to this method is costly, which makes this method of ily reused and can exhibit satisfactory stability. Because of
immobilization expensive. the porosity and increased surface area, the cross-linking of
enzymes to electrospun nanofibers shows a better residual
activity (Park et al. 2013). The advantage of this method is
Cross‑linking that by using suitable stabilizing agents, there is a possibility
to adjust the microenvironment for enzymes through surface
In this method, the biocatalysts are attached using bifunc- complementarity, which in turn increases the stability. This
tional or multi-functional ligands or reagents, which act as method is pertinent to essentially any enzymes, including
linkers (Datta et al. 2013). Since the enzymes behaves as oxidoreductases (cofactor dependent enzymes). However,
their own carrier, this process is also termed as carrier-free there is a disadvantage to conformational changes due to
immobilization (Sheldon 2007a, b). It is also an irrevers- glutaraldehyde addition, which can be reduced by adding
ible method and does not require any support materials to inert proteins like Bovine Serum Albumin (BSA) and gela-
prevent the leakage of enzymes from the substrate (Wang tin (Guisan 2006).
et al. 2008; Honda et al. 2005). The most commonly used
linking agent is glutaraldehyde because they have more Single immobilization versus co‑immobilization
advantages than others, such as being economically afford- of enzymes
able and readily available (Sheldon 2007a, b; Hanefeld et al.
2009). Cross-linking is a pH-dependent method that includes Enzyme immobilization is a technique that enables the recy-
both Schiff’s base formation and Michael-type 1,4 in addi- cling of enzymes, improvement of stability, and enhancing
tion to α, β-unsaturated aldehyde moieties (Migneault et al. activity. However, the immobilization of a single enzyme
2004). Based on the usage of cross-linking enzyme aggre- has some limitations like the deactivation of enzymes by
gate (CLEA) and cross-linking enzyme crystal (CLEC), the products formed, and single targeted pollutant at a time
there are two methods in cross-linking (Sheldon 2011). that leads to high cost and more number of steps to isolate or
for the degradation process of the contaminants (Gustafsson
1. Cross-linking enzyme aggregate et al. 2015). The development of a co-immobilized multi-
enzymatic system is an emerging technology that enables
one to perform multiple reactions instead of a single reaction
• Enzymes are aggregated in precipitants like acetone, at a time as shown in Fig. 3, thereby accumulating undesired
ammonium sulfate, and ethanol. by-products and intermediates be avoided/reduced (Betancor
• A reaction with cross-linkers follows the above step. and Luckarift 2010). For co-immobilization, mostly func-
tionalized nanomaterials/nanocomposites are used.

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Environmental Chemistry Letters

Fig. 3  Single and co-immobilization. Single immobilization: immo- bilization: immobilizing more than one enzyme on the surface of
bilizing a single enzyme on the surface of the immobilization matrix. the immobilization matrix.1. Degradation of the target I—high toxic
1. Degradation of the target I—high toxic compound (specific sub- compound. 2. Degradation of the intermediate product formed takes
strate to the enzyme). 2. There is no degradation of the intermediate place. 3. Degradation of target II takes place
product formed. 3. There is no degradation of the target II. Co-immo-

The co-immobilization of laccase and 2, 2′-azinobis Stability analysis of immobilized enzyme vs free
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) through enzyme
­Cu2+ chelation was the first used method to remove pol-
lutants. It expressed maximal enhanced activity, enzyme Stability is an important criterion in the investigation of immo-
loading (1.7-fold that of free laccase), lower temperature bilized enzymes. The immobilized enzyme’s stability is ana-
and pH sensitivity, and improved storage and thermal stabil- lyzed mostly in measures of thermal stability, pH stability, and
ity. The efficiency in removing pollutants by bisphenol A, storage stability. Various research results have shown that the
indole, and anthracene in water increased by 100%, 70.5%, stability of enzymes has increased. To determine enzymes’
and 93.3%, respectively (Qiu et al. 2020a, 2020b). The co- stability, both free and immobilized enzymes are subjected
immobilization of laccase and (2,2,6,6-Tetramethylpiperi- to the same environmental conditions. While determining
din-1-yl)oxyl (TEMPO) onto amino-functionalized magnetic the stability of the enzymes, a new term “residual activity”
­Fe3O4 nanoparticles showed the maximum decolorization of was used with an initial activity of 100% (Qiu et al. 2020a, b)
acid fuchsin rate of 77.41% and retained above 50% residual and 3-parameter model can be used to determine the stability
activity after eight cycles of operation (Gao et al. 2018a, b). (Arsenault et al. 2011).
The co-immobilization of glucose oxidase with magnetic
graphene oxide showed about 96.6% for the decolorization At ∕Ao = C ∗ exp(−𝛼 ∗ t) + (1 − C) ∗ exp(−𝛽 ∗ t) (1)
process of orange G and was significantly greater than 86.2% where ­At/A0—enzyme activity remaining after time t. α, β—
when used individually. It also showed a high residual activ- Complex expressions of rate constants and C indicates the
ity of 95.2% at the temperature between 42 and 50 °C (Gao weight between the two exponential parts of the equation.
et  al. 2019).  Thus, many research results show that the The values of this model’s parameters were obtained by
removal efficiency was considerably increased. Numerous curve fitting in the plot of the residual enzyme activity versus
studies have shown that co-immobilization increases stabil- time. Even for the same enzyme, based on the immobiliza-
ity, and multi-targeting at a time, which helps in reducing tion matrix, the stability may increase or decrease compara-
treatment steps and costs. tively. Some of the recent research findings of enzyme stability
are summarized in Table 2.

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Table 2  Stability analysis of enzymes

Enzyme Stability Residual activity Immobilization matrix References

Alpha-amylase Thermal stability − Silica coated modified Sohrabi et al. (2014)

nanoparticles
Laccase Thermal stability—after 7 h 70.1% (while free Magnetic Chitosan Nano- Chen et al. (2018)
at 60 °C enzyme—7%) particles
Laccase Storage stability—after 59.1% Magnetic Chitosan Nano- Zappi et al. (2018)
30 days particles
Laccase Storage stability—after 83.5% Nanocomposite Qiu et al. (2019)
30 days
Laccase Storage stability—after 86% Halloysite nanotubes Kadam et al. (2017)
30 days
Laccase Thermal stability—after 3 h 74% (while free enzyme Mesoporous nanospheres Shao et al. (2019)
at 60 °C 35%)
Laccase pH stability—pH 4 90% (while free TiO2 nanoparticles García-Morales et al. (2018)
enzyme—65%)
Laccase Thermal stability—25 to 85% TiO2 nanoparticles García-Morales et al. (2018)
60 °C
Cellulase pH stability—pH 8 20% (while free Magnetic nanoparticles Abraham et al. (2014)
enzyme—0%)
Cellulase pH stability—pH 7 50% (while free PAA nanogel Ahmed et al. (2017)
enzyme—40%)
Cellulase pH stability—pH 8 35% (while free Silica nanoparticles Grewal et al. (2017)
enzyme—10%)
Cellulase pH stability—pH 8 80% (while free Styrene/maleic anhydride Wang et al.2018)
enzymes—14%) copolymer nanoparticles
Manganese Peroxidase Storage stability—after 80% (while free Nanoclay Acevedo et al. (2010)
6 months at 4 °C enzyme—13%)
Laccase Storage stability -after 85% Silica nanoparticles Dehghanifard et al. (2013)
1 month

Accessible nanosubstitutes biological methods are laser ablation, thermolysis, sono-

chemical, and methods like Bacteria, Fungus.
Magnetic nanoparticles The immobilization of enzymes in ­Fe3O4 magnetic nano-
particles follows any one of the following approaches:
Magnetic nanoparticles are well-known for their reusability.
As previously discussed, one of the disadvantages of using 1. Covalent coupling of the enzyme on the surface of the
nanomaterials as immobilization matrices was its recovery coated nanoparticles after the surface functionaliza-
after usage. Nevertheless, the magnetic nanoparticles are tion (Šulek et al. 2010).
easier to be separated after a reaction using a simple mag- 2. Adsorption of the enzyme by non-covalent interactions
netic field (Senthil Kumar et al. 2018b). The hydroxyl group with or without the coating of the nanoparticles (Zhang
at the surface enables the easy functioning and binding of et al. 2008).
enzymes. Low porosity and high mechanical strength reduce 3. Bioaffinity immobilization via fusion tags, which shows
steric hindrance, which is important for enzyme-matrix sta- an affinity toward iron oxide or silica coat over nanopar-
bility (Li et al. 2013). Among different magnetic nanoparti- ticles (Iype et al. 2017).
cles, including alloy-based materials such as ­CoPt3 and FePt, 4. Entrapping enzyme aggregates inside super paramag-
pure metals such as Fe and Co, iron oxides such as ­Fe3O4 netic nanoparticles (Kim et al. 2005).
and γFe2O3, and spinel-type ferromagnet such as M ­ gFe2O4,
­MnFe2O4, and C ­ oFe2O4, ­Fe3O4 is widely used because of its Mesoporous nanoparticles
outstanding attributes, namely biocompatibility and toxic-
free property (Liu et al. 2009). Mesoporous nanoparticles are effective immobilization
Various chemical methods to synthesize magnetic nano- carriers for many enzymes and they have created more
particles include chemical reduction, co-precipitation and interest in the last two decades. They are well-known for
hydrothermal (Siddiqui et al. 2018). Also, the physical and their improved catalytic activity, stability, and reusability.

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Environmental Chemistry Letters

The unique characteristics that differentiate mesoporous by free radicals is also eliminated because of its antioxidant
nanoparticles from other nanoparticles are the fine tuning properties(Lee et al. 2014).
property. This can be achieved by changing the synthesis Immobilization can be done in many ways, including
conditions, thereby defined pores size can be also achieved. non-specific attachment through physical adsorption. The
Mesoporous nanoparticles are available in various forms surface chemistry of graphene-based nanoparticles influ-
like thin films, curved solids, tubes, rods and fibers, mem- ences enzyme immobilization and its activity. Non-covalent
branes, and other monoliths and with the pore sizes rang- interaction between enzymes and nanoparticles may cause
ing from 2-40 nm. Pore interconnectivity is highly desirable leakage of enzymes. Hence, the covalent functionalization
and shows higher mass transfer and diffusion(Linares et al. of graphene-based nanoparticles can be done using ionic liq-
2014). uids (Jiang et al. 2012) or calcium ions (Cazorla et al. 2012).
The immobilization of enzymes in mesoporous nanopar- Covalent immobilization leads to increased enzyme sta-
ticles can be carried out by adsorption (Kim et al. 2011), bility; thereby, we can reduce or eliminate enzymes’ leak-
covalent bonding (Pandya et al. 2005), or cross-linking. age. For covalent immobilization, the use of cross-linking
Sometimes, functionalized mesoporous nanomaterials agents is a widely used approach. For example, 1-ethyl-
enhance stability (Wang et al. 2012). 3-(3-dimethyl aminopropyl) carbodiimide (EDAC) forms
O-acylisourea is highly reactive, and has a tendency to form
Carbon‑based nanoparticles covalent bonds with free amine groups of enzymes (Gao and
Kyratzis 2008).
Graphene‑based nanoparticles
Carbon nanotubes
Graphene and graphene oxide are well-known for their char-
acteristics, notably biodegradability, thermal and chemical Carbon nanotubes are hollow cylinders of carbon atoms in
stability, high surface area, pore-volume, and their unique an arrangement of periodic hexagons. They are like rolled-
characteristic occurrence of various functional groups, up layers of graphene that exhibit similar properties to gra-
which enable the researchers/scientists to immobilize phene but in one-dimensional tubular geometry rather than
enzymes without the use of cross-linking agents (Balandin two-dimensional planar geometry, and have a diameter size
et al. 2008; Lee et al. 2008). Figure 4 shows the structure in the range of nanoscale (Georgakilas et al. 2015). Carbon
of graphene. Hydroxyl, carboxylic, and epoxide groups are nanotubes can be constructed in two main forms: single-
some of the functional groups that are present in graphene. walled carbon nanotubes (SWCNTs) and multi-walled car-
These groups enable strong interaction between enzyme and bon nanotubes (MWCNTs). As carbon nanotubes are formed
graphene/graphene oxide without a cross-linking agent and from graphene sheets, they also possess characteristics like
functional group modification (Zhang et al. 2016). All these chemical and thermal stability, high tensile strength and
features make graphene-based nanoparticles an efficient elasticity, high conductivity as well as biocompatibility
immobilization matrix for enzymes. Enzyme inactivation (Bianco et al. 2011), which makes carbon nanotubes an effi-
cient immobilization matrix.

Single‑walled carbon nanotubes

Single-walled carbon nanotubes are rolled up into a single

layer of graphene. There are two standard types of single-
walled carbon nanotubes depending on graphene sheets roll-
ing based on chirality: "zigzag" structure and "armchair"
structure. The third non-standard type of structure is also
available. The diameter of carbon nanotubes varies from a
few nanometers (Saifuddin et al. 2013).

Multi‑walled carbon nanotubes

Multi-walled carbon nanotubes are composed of several

concentric tubes of graphene fitted as one inside the others.
The diameter of carbon nanotubes varies from several tens
Fig. 4  Graphene is a two-dimensional carbon allotrope,  composed of of nanometers. Double-walled carbon nanotubes (DWCNTs)
carbon atoms positioned in a hexagonal design are the simplest example of MWCNTs. They combine the

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outstanding properties of SWCNTs and thus, they have sev- 2015). Eventually, functional modifications can be made for
eral advantages over SWCNTs (Jorio et al. 2008). They are the covalent attachment of enzymes, with the help of some
desirable for easier dispersibility and low cost. cross-linking agents or binders, as mentioned in the func-
For the immobilization of enzymes on carbon nanotubes, tional modification of graphene-based nanoparticles.
non-covalent interactions are considered as a promising
approach compared to covalent methods, as it preserves
the conformational structure of enzymes. In the non-cova- Pollutants
lent mode of immobilization, immobilization can be done
by:i) direct physical adsorption (Gómez et al. 2005), (ii) Due to the industrial revolution, the organic pollutants have
adsorption using biomolecules, polymers, or surfactants. considerably increased and there is a huge need to remove
For example, the immobilization of glucose oxidase on such organic compounds for the efficient wastewater treat-
the surface of SWCNTs can be positively charged by coat- ment. There are several sources for such pollutants, e.g.,
ing poly (sodium4-styrenesulfonate) combined with ionic industrial compounds-polycyclic aromatic hydrocarbons
liquids (Gómez et al. 2005; Wu et al. 2009). Sometimes, (Borji et al. 2020), volatile organic compounds, and color-
biomolecules (Lee et al. 2010) and surfactants (Gao et al. ants. (Das et al. 2020). Organic pollutants can also be used
2016) like Triton-X are also preferred. for recovery of value added products (Selvakumar et al.
The covalent immobilization of enzymes can also be done 2020) and the hazardous organic pollutants could be trans-
by direct linking (Feng and Ji 2011) or by using linking formed into less toxic or non-toxic ones after treatment with
agents, for example, 1-pyrene butanoic acid succinimidyl immobilized enzymes. The efficiency increases when the
ester is used as linking agent for horseradish peroxidase immobilization is held on nanoparticles.
immobilization and aminopyrene is used as linking agent
for laccase immobilization(Kim et al. 2009). Pesticides

Ceramic‑based nanoparticles Pesticides are the unavoidable part of modern agriculture.

These pesticides remain in the soil for years and leach into
Because of thermal stability, resistance to pressure, solvents, groundwater. The removal of such pesticides is essential for
and higher mechanical strength, ceramic materials gain more avoiding toxic revolution. The pesticides include all types
attention and are used in many fields (Ota et al. 2007). The of insecticides and herbicides, which are harmful to the
word “ceramic” indicates solid, inorganic, and nonmetallic environment (Senthil Kumar et al. 2018a; Christopher et al.
materials. The raw materials, method of preparation, and 2020; Rathi et al. 2020). There are many technologies for
its condition greatly impact the properties of the final prod- this removal but in this article, let us discuss the usage of
uct like mechanical strength, porosity, and rigidity (Sigur- enzymes immobilized on nanoparticles.
dardóttir et al. 2018). Thereby, choosing an apt method of Chlorpyrifos [O,O-diethyl O(3,5,6-trichloro-2pyridinyl
preparation, raw material, and conditions, different ceramic phosphorothioate] is a commonly utilized insecticide. To
materials can be obtained. They are usually composed of degrade this insecticide, the magnetic iron nanoparticles
nitrides such as aluminum and silicon nitrides. The composi- were prepared using the co-precipitation method. The pro-
tion may also include aluminosilicates of montmorillonite, duced magnetic nanoparticles were layered with chitosan, by
kaolinite, and bentonitexides of silica, alumina, titania and reverse-phase suspension method, followed by the function-
zirconia and carbides of silicon and boron.. They can be alization of the particles using glutaraldehyde solution. For
used in pure form or composites or hybrid forms by mix- immobilizing laccase enzyme in the functionalized nano-
ing with other polymers. Besides different compositions, particle, the chitosan-layered nanoparticles were activated
many various forms of ceramic materials are also avail- using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide
able. These are available in nanostructures like nanofibers (EDAC). It was concluded that the laccase immobilized on
(García-Bordejé et al. 2007), nanotubes (Wang et al. 2009), magnetic iron nanoparticles was highly potent in degrading
nanosheets, nanoflowers (Gao et  al. 2017), nanoporous greater than 99% chlorpyrifos within 12 h at a pH of 7 and
membranes (Kjellander et al. 2014), and in other forms 60 °C. Also, in the comprehensive degradation percentage,
like microbeads, foams, etc. The hydroxyl-functional group 32.3% of chlorpyrifos removal was contributed by MNPs
present on the surface of the ceramic nanomaterials favors while ENPs caused 58.8% chlorpyrifos degradation (Das
enzyme adsorption. et al. 2017).
Enzyme immobilization can be done by (a) physical The fungal laccases, or multi-copper oxidases, were uti-
adsorption due to weak nonspecific forces  (Facin et  al. lized as catalysts to transform organochlorine pesticides
2019), (b) entrapment and encapsulation, (c) covalent bond- (Zhao et al. 2010). Fungal laccases are efficient degraders of
ing (Eldin et al. 2011), and (d) cross-linking (Mohamad et al. chlorphen (CP) and dichlorophen (DCP) in water (Shi et al.

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2016). Fungal laccase can be immobilized on nanostructured and we discuss the usage of the immobilized enzyme on
silicon foam for the maximum transformation of dichloro- nanoparticles for dye removal (Darwesh et al. 2019).
phen. The internal pore size of MSU-F imposes limitations Laccase enzymes can be immobilized in titania nano-
on the denaturation of proteins, and the associated crowding particles, which results in immobilized laccase in titania
effect within the nanostructure (Vidal-Limon et al. 2018). nanoparticles(ILTNs). They were prepared with the help
Prolidases are said to have biodegradation capacity of 3 amino propyl triethoxysilane and glutaraldehyde.
for toxic organophosphorus (OP) substances(Wang et al. These immobilized enzymes were capable of degrading
2019). Using silica-coated magnetic nanoparticles (SiM- 3 anionic dyes, namely Direct Red31 (DR31), Acid Blue
NPs) with isocyanatopropyltriethoxysilane makes it a 92 (AB92), and Direct Green 6 (DG6) from aqueous solu-
metal-chelating ligand so that the His6-tagged Escherichia tions, both individually and in binary systems. It is also
coli prolidase(His6-EcPepQ) can be immobilized.His6- necessary to consider the concentration of ILTNs, time,
EcPepQ@NiNTASiMNPs evidently has greater activity and pH, which are the essential factors in the decoloriza-
and stability than free enzymes, which can also hydrolyze tion of dyes. ILTNs with low dose of 0.1 to 0.3 g/L were
the two individual organophosphorus compounds, dime- enough to complete the decolorization of the dyes within
thyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl 20 min (Mohajershojaei et al. 2015).
p-nitrophenyl phosphate (ethyl paraoxon).Some of the exam- It is studied that magnetic graphene oxide (MGO)
ples of enzymes along with the immobilization matrix used nanomaterials can be prepared by covalently binding the
for the removal of certain pesticides are discussed in Table 3. amino ­Fe3O4 nanoparticles onto the surface of graphene
Table 3 explains more about the immobilized enzymes oxide (GO). This acts as an effective carrier for immo-
in nanoparticles which are used to remove pesticides, herbi- bilizing laccase. The MGO-laccase disclosed more mag-
cides, and insecticides. netic reactions and manifested their reclaimable property
after magnetic separation when compared to free laccase.
Dye Immobilization was done by covalently binding laccase
and animo ­Fe3O4 nanoparticles onto the GO succeeding
Dye waste, being non-biodegradable, constitutes the major the activation of graphene oxide by 1-ethyl-3-(3-dimeth-
pollutant groups (Renita et al. 2019; Pavithra et al. 2019). ylaminopropyl) carbodiimide (EDAC) and N-hydroxy
They are the most common effluent in the pharmaceutical, sulfosuccinimide (NHS) acting as the bifunctional cross-
chemical, and textile industries. The mutagenic character linking factors. Such MGO-laccases were used in the
and carcinogenicity of dyes are dreadful. Dyes abide in the decolorization processes involved in dyes, which include
environment for a long period due to their higher thermal the several dyes, namely crystal violet (CV), malachite
and photo stability to resist biodegradation. The removal green (MG), and brilliant green (BG). The decolorization
of dyes from wastewater can be done in various processes,

Table 3  Pesticides removal using enzyme immobilization

Enzyme Immobilization matrix Pesticides Removal efficiency References

Glycerophosphodiesterase Magnetic nanoparticles Organophosphate pesticide – Daumann et al. (2014)

(GpdQ)
Laccase Magnetic nanoparticles Chlorpyrifos – Srinivasan et al. (2020)
Laccase Iron oxide nanoparticles Chlorpyrifos 99% Das et al. (2020)
Laccase Mesoporous nanostructured Dichlorophen – Vidal-Limon et al. (2018)
silicon foam
Laccase Tubular mesoporous silica Methoxychlor 45.6% Yang et al. (2016)
containing ultrasmall super-
paramagnetic iron oxide
nanoparticles
Chloroperoxidase Halloysite nanotubes Isoproturon – Fan et al. (2018)
Organophosphorus hydrolase Poly Cyclodextrin-based Nano- Organophosphate – Moon et al. (2019)
particles
Organophosphate hydrolases Mesoporous silica nanopar- Paraoxon – El-Boubbou et al. (2012)
ticles
Carboxylesterase Mesoporous silica nanopar- Malathion and Diethofencarb 95 and 92% Diao et al. (2013)
ticles
Laccase Cellulose nanofibers Trifluralin 85% Bansal et al. (2018)

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rate was also increased to 94.7% of CV, 95.6% of MG, and Phenolic compounds
91.4% of BG, respectively (Chen et al. 2017).
The enzymatic extracts of two fungi isolates Pleuro- Phenolic compounds are observed in the effluents of cer-
tusostreatus (PLO9) and Ganodermalucidum (GRM117) tain industries such as oil refining, petrochemicals, pharma-
containing lignin peroxidase (LiP) were immobilized on ceuticals, resin manufacturing, plastics, paint, pulp, coking
carbon nanotubes. The stability, efficiency of being a operation, paper, and wood products industries (Sun et al.
catalyst, and the capacity of being reused in dye decol- 2015; Kazemi et al. 2014; Mohammadi et al. 2015). Bisphe-
orization processes were found to be increased(Oliveira nol A (BPA) (Khazaali et al. 2014), chlorophenols (CPs),
et al. 2018). and phenolic endocrine-disrupting compounds (Zhang and
Ginger peroxidase was immobilized on amino-func- Li 2014) are common derivatives of phenols. Additionally,
tionalized silica-coated titanium dioxide nanocomposite. phenolic substances are major pollutants with higher toxic-
Immobilized peroxidase exhibited higher activity and ity levels even at low concentration levels, thus necessitat-
thermostability than free peroxidase. Immobilized per- ing its removal from the wastewater. Some of the examples
oxidase had a greater potential for removing acid yellow of enzymes along with the immobilization matrix used for
42 dyes in a stirred batch process by decolorizing 90% the removal of certain phenolic compounds are discussed
of the dye in 1.5 h, while free enzymes decolorized only in Table 4.
69% of the dyes in the same stipulated period. In addition, The immobilization of laccase was done on various car-
even after its repeated use for 6 times, the immobilized bon nanomaterials like MWCNTs, O-MWCNTs, graphene
peroxidase still exhibited about 62% of its dye decoloriza- oxide, and fullerene (C60) to act as a biocatalyst and degrade
tion activity (Ali et al. 2017). Some other findings of the bisphenol A (BPA), and catechol phenolic contaminants.
removal of certain dyes using nanoimmobilized enzymes The immobilization was done using physical adsorption
are discussed in Table 4. rather than covalent binding. The immobilized enzymes
also reduced the reaction rate: the reaction rates of graphene

Table 4  Dye and phenolic compound removal using enzyme immobilization

Enzyme Immobilization matrix Target molecules Removal efficiency References

Horseradish Peroxidase ZnO nanowires/macroporous Acid Blue 113, and Acid 95.4% and 90.3% Sun et al. (2017)
­SiO2 composite Black 10 BX
Horseradish Peroxidase Carbon Nanosphere 2,4-chlorophenols, 4‐meth- 95%, 99.3% and 51.7% Lu et al. (2017)
oxyphenol, and Bisphenol
A
Horseradish Peroxidase Reduced Graphene Oxide Phenols 100% Besharati Vineh et al. (2018)
Lignin Peroxidase Carbon Nanotubes Remazol Brilliant Blue R 40% Oliveira et al. (2018)
Laccase Chitosan Nanoparticles Congo red 98% Sadighi and Faramarzi (2013)
Laccase Fe3O4/SiO2 nanoparticles Procion Red MX-5B 96% Dai et al. (2016)
Chloroperoxidase Fe3O4 magnetic nanoparticles Aniline blue 90% Cui et al. (2015)
Laccase Magnetic poly(p-phenylene- Blue 19 80% Liu et al. (2016)
diamine) (PpPD) nanocom-
posite
Peroxidase Polypyrrole-cellulose- Blue 4 99% Ali et al. (2018)
graphene Oxide Nanocom-
posite
Laccase Metal-chelated Chitosan Phenol 96% Alver and Metin (2017)
Based Nanoparticles
Laccase Fe3O4/SiO2 nanoparticles Procion Red MX-5B, and 80% Wang et al. (2013)
azophloxine
Laccase Magnetic Bimodal Phenol and p-chlorophenol 78% and 84% Liu et al. (2012)
Mesoporous Carbon
Laccase Magnetic Nanoparticles Phenol,4-chlorophenol, and 86.1%, 93.6% and 100% Qiu et al. (2020a, 2020b)
2,4-dichlorophenol
Laccase ZnO and ­MnO2 Alizarin Red S dye 95% and 85% Rani et al. (2017)
Laccase Electrospun Chitosan/Poly 2,4-dichlorophenol 87.6% Xu et al. (2013)
(Vinyl Alcohol) Composite
Nanofibrous Membranes

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oxide-laccase, which are conjugated with bisphenol or cat- leads to the formation of so-called ‘superbugs’ which are
echol substrates were 10.28% and 12.33%, accordingly as dreadful in various aspects and necessitates their removal
compared to free enzymes (Pang et al. 2015). from the wastewater (Xu et al. 2015).
By the arrangement of chitosan onto the surface of hal- Amino functionalized magnetic nanoparticles are used to
loysite (natural nanotubular aluminosilicate), Chitosan–hal- immobilize β-lactamase through cross-linking in the cova-
loysite hybrid-nanotubes were prepared. Such nanotubes lent binding method. The immobilized enzyme was efficient
had an excellent capability to immobilize horseradish per- in the degradation of β-lactam antibiotics. Immobilized
oxidase. The immobilization was carried out through the β-lactamase was subjected to different ranges of tempera-
process of cross-linking with the help of glutaraldehyde. ture and pH for the degradation of penicillin G. When com-
The maximum enzyme loading capacity was achieved by pared to the free enzyme, storage and thermostability of the
about 21.5 mg/g, higher than 3.1 mg/g of raw halloysite. β-lactamase enzyme was increased. Also, the efficiency of
The horseradish peroxidase (HRP) immobilized on HNT/ degradation of 5 mg ­L−1 penicillin G solution was greater
CTS retained the activity in a wider range and it also pos- than 95%. (Gao et al. 2018a, b).
sesses very good storage stability. Overall, the immobilized Fe3O4 nanoparticles were prepared through the chemical
horseradish peroxidase was used as a catalyst for the removal co-precipitation method which is then functionalized with
of phenol from wastewater and it was also identified that the 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, and
removal efficiency is over 80.0%, that too in a shorter dura- Con A. Then, the laccase from Echinodontiumtaxodii was
tion (Zhai et al. 2013). immobilized on concanavalin A-activated ­Fe3O4 nanopar-
Modified magnetic iron oxide nanoparticles carrying ticles (GAMNs-Con A) based on the analysis of the sur-
tyrosinase (tyrosinase-MNPs) which acts as a magnetic face of laccase by affinity adsorption. Activity recovery and
nano-bio-catalyst were utilized to treat wastewater, which enzyme loading for these nanoparticles were higher than the
contains phenol pollutants. In this, the tyrosinase was immo- capability of conventional covalent binding. The substrate
bilized onto cyanuric chloride-functionalized magnetic nan- affinity of immobilized laccase was also higher in compari-
oparticles. The phenol degradation was more than 70% by son to free enzymes. In GAMNs-Con A-L, syringic acid
immobilized tyrosinase when the substrate having a greater is responsible for the fastest elimination efficiency of SAs
concentration of 2500 mg/L was applied to remove phenol. among S-type compounds. As a result, the substances were
In this, the phenol degradation is 100% even after the reuse removed entirely after an incubation duration of 5 min and
cycle for the third time and even about 55% succeeding in the rate of removal of SAs was also higher (Shi et al. 2014).
the seventh cycle. Such immobilized tyrosinase was also Laccase was immobilized either by cross-linked enzyme
proficient in degrading the phenol that is dissolved in water aggregates (CLEAs) or magnetic cross-linked enzyme
samples up to 78% after an incubation period of 60 min. aggregates (mCLEAs). Under favorable conditions, the
Hence fabricated nano-bio catalysts are said to be promising capacity of removal of native laccase by the Diclofenac was
for micropollutant removal (Abdollahi et al. 2018). 11.5 ± 0.3 μg DCF/g laccase, by CLEAs was 15.6 ± 0.4 μg
Graphene oxide/Fe3O4 (GO/Fe3O4) nanoparticles were DCF/g laccase, and by mCLEAs, it was 13.6 ± 0.4  μg
prepared by an ultrasonic-assisted reverse co-precipitation DCF/g laccase. These outcomes showed that the removal
method followed by the immobilization of horseradish per- capacity was greater in immobilized enzymes than free
oxidase onto GO/Fe3O4 with 1-ethyl-3-(3-dimethylamino- enzymes (Primožič et al. 2020).
propyl) carbodiimide (EDAC) as a cross-linking agent in
the covalent binding method. The immobilized enzyme was
efficient to remove 95% of phenol from the aqueous solu- Mechanism of the removal of pollutants
tions. The horseradish peroxidase immobilized on GO/Fe3O4
composite was easily separated within a magnetic field from Many research papers describe the performance of nano-
the reaction solution and can be reused after that. The system materials in the field of environmental pollution reduc-
was able to catalyze the reduction of ­H2O2 to remove phenol tion. The process of removal of toxic pollutants are said to
and 2,4-dichlorophenol (2,4-DCP)(Chang et al. 2016). be the process of filtration (Jurecska et al. 2014), photo-
catalytic activity (Wu et al. 2020; Saravanan et al. 2020),
Antibiotics and adsorption (Tanzifi et al. 2020), enzymatic degra-
dation/biodegradation (Besharatsi Vineh et  al. 2018).
Antibiotics in the marine environment have the potential to Nevertheless, when we talk about enzyme-loaded nano-
create an impact on the water quality and health of human particles, the name itself depicts the principle that the
lives. As the global usage of antibiotics increases, there is a enzymatic action degrades the pollutants. For this, we use
risk of the emergence of new drug-resistant microorganisms specific enzymes. As discussed earlier, either one enzyme
and their interaction with bacteria present in the water. This can be immobilized (single immobilization) and used at

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Table 5  Removal efficiency by degradation, adsorption and combined degradation and adsorption

Nanomaterial Enzyme Pollutant Removal efficiency References
Degradation Adsorption Total
(%) (%) (%)

Multi-walled carbon nanotubes Laccase Bisphenol A 71 9 80 Pang et al. (2015)

Epoxy-functionalized silica Laccase Phenolic compounds 70 10 80 Mohammadi et al. (2018)
Alumina pellets Laccase Reactive Black 5 90 10 100 Osma et al. (2010)
Hollow Mesoporous Carbon Laccase Tetracycline 55 77 100 Shao et al. (2019)
Spheres
Magnetic Chitosan Manganese peroxidase Dyes (Textile effluent) 90 20 97 Bilal et al. (2016)
Beads
Cross-linked Chitosan Beads Laccase Sulfur Blue 20 65 85 Nguyen et al. (2016)
Graphene oxide nanofibers Laccase Catechol 60 35 38 Wang et al. (2014)
Silica nanoparticles Laccase Triclosan 40 25 65 Xu et al. (2014)
Silica nanoparticles Horseradish peroxidase Paracetamol 84 15 98 Xu et al. (2015)

a time or two or more enzymes can be immobilized (co- Conclusion

immobilization) and used.
The biodegradation of hazardous compounds using There are currently multiple ways to treat wastewater. One
immobilized enzymes is commonly reported to be very such process is the immobilization technique for the removal
efficient and selective, but they have few limitations. and degradation of toxic pollutants. This review comprehen-
The limitations are (a) Enzymes show high activity only sively discusses the overview of the nanoparticles and their
within a narrow range of conditions such as temperature role in immobilization techniques to remove toxic contami-
and pH (Al-Khalid and El-Naas 2012), (b) Incomplete nants in wastewater. The immobilization process has been
deterioration or elimination of pollutants, and (c) Inacti- used for enhancing enzyme activity and stability in aqueous
vation of enzymes by the products formed via blocking and non-aqueous media. The selection of carrier materials
the active sites (Gasser et al. 2014). is an essential factor to be considered in the immobilization
Nevertheless, many attempts are currently made to technique. Due to their small size and large surface area,
combine biodegradation and adsorption methods to nanoparticles and their derivative matrices are a versatile
develop more efficient and economically beneficial tool for enzyme stabilization, increasing the efficiency of
strategies for the degradation of hazardous compounds. organic and inorganic pollutants removal. Various studies on
Adsorption is less sensitive to changes in environmen- toxic pollutant removal by using the immobilized technique
tal conditions. The adsorption process is used when the have been thoroughly discussed. It can be concluded that the
biodegradation of a solution with a high concentration of nanoparticles immobilized enzymes are effective for remov-
toxic compounds is carried out. However, enzymatic deg- ing toxic pollutants from the wastewater. Today, commer-
radation is highly efficient in lower concentrations. When cially immobilized enzymes have revealed high efficiency.
the pollutant concentration is high, its degradation time is
also high, which increases the cost of the process. Simul-
taneous enzymatic degradation and adsorption enhance Funding  Authors declares that this work is not financially supported
the removal of hazardous compounds and allow attaining by any funding agencies.
high removal efficiency quickly. It not only adsorbs toxic
pollutants but also removes products of degradation (Ali Compliance with ethical standards 
et al. 2012), even though the products are not hazard-
Conflict of interest  The Authors declare that they have no conflict of
ous. It helps in maintaining enzyme activity by eliminat- interest.
ing products that block the active sites of enzymes. This
can be done by using appropriate sorbents. The pollutant
removal efficiency of some enzymes using degradation References
method and adsorptions are discussed in Table 5
Abdollahi K, Yazdani F, Panahi R, Mokhtarani B (2018) Biotransfor-
mation of phenol in synthetic wastewater using the functionalized

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Environmental Chemistry Letters

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