Accepted Manuscript

Magnetic nanoparticles as versatile carriers for enzymes

immobilization: A review

Muhammad Bilal, Yuping Zhao, Tahir Rasheed, Hafiz M.N. Iqbal

PII: S0141-8130(18)33760-7
DOI: doi:10.1016/j.ijbiomac.2018.09.025
Reference: BIOMAC 10460
To appear in: International Journal of Biological Macromolecules
Received date: 22 July 2018
Revised date: 2 September 2018
Accepted date: 5 September 2018

Please cite this article as: Muhammad Bilal, Yuping Zhao, Tahir Rasheed, Hafiz M.N.
Iqbal , Magnetic nanoparticles as versatile carriers for enzymes immobilization: A review.
Biomac (2018), doi:10.1016/j.ijbiomac.2018.09.025

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 ACCEPTED MANUSCRIPT

Magnetic nanoparticles as versatile carriers for enzymes immobilization: A review

Muhammad Bilal*a, Yuping Zhaoa, Tahir Rasheedb, and Hafiz M.N. Iqbal*c
aSchool of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian
223003, China; bSchool of Chemistry and Chemical Engineering, State Key Laboratory
of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China;
cTecnologico de Monterrey, School of Engineering and Sciences, Campus Monterrey,
Ave. Eugenio Garza Sada 2501, Monterrey, N.L., CP 64849, Mexico; Corresponding

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author emails: bilaluaf@sjtu.edu.cn (M. Bilal); hafiz.iqbal@itesm.mx (H.M.N. Iqbal)

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Abstract
Enzymes are highly efficient biocatalysts and widely employed in biotechnological

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sectors. However, lack of (re)-purification and efficient recovery of enzymes are among
the most critical and challenging aspects, which render them enormously expensive for
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industrial exploitability. Aiming to tackle these challenges, magnetic nanoparticles
(MNPs) have gained a special place as versatile carriers and supporting matrices for
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immobilization purposes, owing to the exceptional properties of MNPs, such as large

surface area, large surface-to-volume ratio, and mobility and high mass transference.
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More importantly, they can also be easily separated and recovered by applying an
external magnetic field. Apart from their biocompatible micro-environment, the utilization
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of such MNPs represents a noteworthy green chemistry approach, since it lengthens

the biocatalyst lifetime through multiple recovery cycles. According to the literature
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evidence, various modification and/or functionalization approaches have been

developed to produce MNPs for the effective immobilization of a broad variety of
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industrially important enzymes and biomolecules with improved characteristics.

Enzymes immobilized on MNPs displayed a wide-working pH and temperature range,
as well as, improved thermal and storage stabilities than that of their pristine
counterparts. Co-immobilization of multi-enzymes could also be accomplished through
nanoparticle-based approaches. This review presents an updated outlook on the
development and characterization of MNPs, in particular, iron-based MNPs-derived
nano-constructs as support materials for enzyme immobilization.

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Keywords: Enzyme immobilization; Green chemistry; Magnetic nanoparticles;

Supporting materials; Surface functionalization

1. Introduction
Enzymes are very efficient (bio)catalysts that catalyze numerous biochemical and
chemical reactions under mild reaction conditions such as ambient temperature,
physiological pH, and aqueous environment, etc. They carry out very precise reactions

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owing to their exceptional chemo-, regio- or stereospecificity and selectivity [1-5]. Due to

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their easy and green synthesis, substrate specificity and environmental friendliness,

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enzymes have been considered as potential candidates for diverse applications in
organic syntheses [6, 7], healthcare and pharmaceuticals [8], and chemicals and foods

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[9-11]. Nevertheless, all these desired enzymes characteristics and their widespread
industrial perspective are seriously hampered by low operational stability and marginal
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lifetime [12]. Among the various modification approaches including protein engineering,
use of additives and immobilization, the enzyme immobilization is a preferred strategy in
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overcoming the above-stated obstacles and to improve the enzyme catalytic stability for
different applied purposes [4, 5, 13-22]. Despite an array of various emerging support
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materials used for the immobilization, such as chitosan, graphene oxide, or

polyurethane foam, the exploitation of novel immobilization matrices and technologies
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are still appealing substantial attention.

The boom of nanosciences has encouraged a profound interest in properties, which
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depend on the particle size. Magnetic nanoparticles, for instance, display their greatest
performance at typical sizes ranges from 10–20 nm due to the emergence of
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superparamagnetism [23, 24]. Such features make them with a fast response to applied
magnetic fields. Also, these nanoparticles demonstrate huge specific surface area, large
surface-to-volume ratio, easy separation under external magnetic fields, and high mass
transference, perfect characteristics for support in catalytic systems [25, 26]. In enzyme
immobilization, the adsorption capacity of the enzyme on a surface depends on the
activation of chemical bonds on the surface of both nanoparticle and protein [27].
Nonporous materials did not exhibit diffusion limitations for immobilizing enzymes, but
the enzyme loading per unit mass of carrier support is rather low. On the other hand,

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these materials can afford higher loading of the enzyme, however, suffer from
considerable diffusional restrictions for the substrates. Magnetite is the most widely
pursued magnetic support for immobilizing biomolecules/proteins owing to a greater
saturation magnetization and susceptibility. In contrary, magnetite with diameters lesser
than 8 nm is quite challenging to stabilize against oxidation, generating mixtures of
oxides with reduced crystallinity and magnetization response [28]. Since the bare/naked
iron magnetic nanoparticles often exhibit high reactivity and easily degraded under

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certain environment, leading to poor dispersity and stability. For this, several

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modification approaches have been described in the literature to generate

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biocompatible iron magnetic nanoparticles for efficient immobilization of enzymes [25].
Herein, recent and up-to-date literature on the synthesis and characterization of iron-

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based MNPs for the enzyme immobilization is summarized. Following a brief
introduction, the first half focuses on the unique properties and surface modification
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and/or functionalization of MNPs. Enzyme immobilization on various MNPs forms such
as silica-coated MNPs, organic polymer-modified MNPs, mesoporous material-modified
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MNPs metal-organic frameworks (MOFs)-based MNPs is also discussed with suitable

examples. Lastly, economic evolution and application perspectives are given with
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concluding remarks and future outlook.

2. Magnetic nanoparticles and their properties
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After biocatalytic processes, the separation of immobilized biocatalysts from the

enzymatic reaction mixture is one of the key challenges that need to be overcome for
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immobilization. At this juncture, associating biomolecules with magnetic nanoparticles

(MNPs) provides a facile separation of the biocatalytic system by the use of an external
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magnetic field. In recent decades, MNPs have attracted a great deal of attention as
promising support carriers in enzymes immobilization due to the large surface area and
the presence of hydroxyl groups on their surface which enables their easy
functionalization and strong binding of the enzyme molecule. Low porosity and
exceptional mechanical stability are the characteristic features, which reduce steric
hindrances and are important for the development of a stabilized enzyme-matrix
catalytic system [29]. All these features led to a substantial improvement in loading
capacity of enzymes or biomolecules. The magnetic properties of these support

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materials can promise the facile separation of encapsulated enzymes from reaction
media, and therefore rapidly terminating the enzymatic reactions and recovering the
enzymes for their continual uses. In this way, the tedious centrifugation process can be
circumvented, which greatly simplifies the enzyme immobilization and recovery
procedures [26]. Table 1 summarizes the use of various magnetic nanoparticles as
support materials for the immobilization of a great variety of industrially important
enzymes and their biotechnological applications.

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Amongst the different MNPs including iron oxide (Fe3O4 and γ-Fe2O3), alloy-based

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(CoPt3 and FePt), pure metal (Fe and Co), and spinel-type ferromagnet (MgFe2O4,

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MnFe2O4, and CoFe2O4) MNPs, Fe3O4 NPs are the most widely pursued for the
immobilization of enzymes owing to their remarkable advantages of biocompatibility and

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non-toxicity [52]. Recently, a great variety of enzymes oxidoreductases, hydrolases or
transferases have been immobilized/insolubilized on the surface of functionalized MNPs
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to develop stable catalytic systems with an easy separation and high repeatability
characteristics [24]. For example, Atacan et al. [53] used gallic acid pre‐treated MNPs
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for the covalent attachment of trypsin enzyme. The resulting MNPs‐trypsin biocatalyst
was shown to degrade bovine serum albumin (BSA) with great catalytic efficiency in the
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wide working environment. In another study, original MNPs-adsorbed glucose oxidase

(GO) enzyme retained more than 90% of its initial activity after 15 continuous catalytic
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cycles for the decolorization of acid yellow 12 dye [54]. Mehrasbi et al. [55] reported that
immobilized lipase on MNPs functionalized with 3‐glycidoxypropyltrimethoxysilane
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efficiently catalyzed the production of biodiesel from waste cooking oil. Interestingly, the
modified lipase system preserved 100% of its original activity even after repeated use of
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six reaction cycles. Li et al. [35] developed MNPs and co-immobilized enzyme by metal
coordinated hydrogel nanofibers for the immobilization of lipase from Candida rugosa
(Figure 1). In comparison with free enzyme, the relative activity of magnetic iron oxide
nanoparticles-anchored lipase was 8-fold and 1-fold increased at pH 10.0 and 50 °C,
respectively. Further, nanoparticles immobilized enzyme exhibited long-term storage
stability (nearly 80% relative activity after 13 days of storage) indicating metal-
nucleotide nanofibers as a biocompatible support material for enzyme immobilization to
improve their stability features.

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Zhou et al. [56] fabricated a convenient and an efficient Fe 3O4/PMG/IDA-Ni2+

nanoparticles-based support to immobilize β-glucosidase amplified from Coptotermes
formosanus Shiraki. The immobilized biocatalyst showed excellent catalytic activity
and stability compared with free derivative and retained more than 65% of the original
activity after recycling for many times. Horseradish peroxidase (HRP) was physically
adsorbed on pristine iron magnetic nanoparticles and characterized by scanning
electron microscopy (SEM), FT-IR spectroscopy, and energy dispersive X-ray. The

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support-mediated HRP catalyst maintained 55% of its initial activity after consecutive

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uses for 10 times and was more thermostable than non-immobilized form. Various

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substrates oxidizing capacities were improved by the immobilized HRP in contrast with
the soluble enzyme. Of most recent, Hosseini et al. [57] developed a magnetic

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nanocomposite by entrapping Fe3O4 nanoparticles into the cross-linked ionic
liquid/epoxy type polymer and used for covalent attachment of cellulase enzyme.
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Notably, the catalytic performance and stability of the carrier-bound enzyme were
significantly improved along with reusability compared to pristine cellulase.
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3. Surface modification/functionalization of MNPs

Several methods have been attempted to fabricate Fe3O4 nanoparticles (Table 2) [25].
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Among them, chemical co-precipitation, micro-emulsion, thermal decomposition and

solvothermal are considered to the typical synthetic routes [26]. Generally, Fe3O4
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nanoparticles tend to aggregate due to their high surface energy triggered by the large
specific surface area. Additionally, Fe3O4 nanoparticles possess high chemical activity
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and are easily oxidized in the air leading to loss of magnetism and dispersibility [25, 58].
These factors render the magnetic separation less effective resulting in the
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incompetence of naked Fe3O4 nanoparticles for direct attachment of enzymes. In this

context, surface functionalization might assist in preventing the aggregation and
oxidation of Fe3O4 nanoparticles.
3.1 Enzyme immobilization on silica-coated MNPs
Silica coating, in which silica shells formed on the surfaces of magnetic cores, is a
distinctive method for the functionalization/modification of Fe 3O4 nanoparticles. The
development of silica shells improves the chemical stability by protecting the magnetic
cores both from aggregation and oxidation. It can also enhance the hydrophilicity and

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biocompatibility characteristics. Introduction of substantial silanol groups on the

surfaces of magnetic cores after modification provides the underlying basis for further
modification with functional reagents for enzymes immobilization. Fe 3O4 nanoparticles
can be directly coated with silica shells by a well-known sol-gel process [59], in which
tetraethoxysilane is hydrolyzed under alkaline conditions, provoking to develop silica
shells on the surfaces of magnetic cores to obtain the core-shell Fe3O4@SiO2
nanoparticles. The core-shell Fe3O4@SiO2 nanoparticles are typically first functionalized

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with amino silane [60], or epoxy silane coupling agents [61] to introduce amino or epoxy

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groups on the surfaces of support matrices, respectively, for efficient enzymes

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attachment. Subsequently, enzymes can be immobilized on the surface of amino-
functionalized Fe3O4@SiO2 nanoparticles based on Schiff base reactions with the help

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of glutaraldehyde; whereas enzymes can be directly immobilized on epoxy-
functionalized support matrices through the interactions between an amino group of
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enzyme and epoxy groups of support. In contrast with amino-functionalized
Fe3O4@SiO2 nanoparticles, no additional coupling is involved for enzymes
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immobilization on epoxy-functionalized Fe3O4@SiO2 nanoparticles. Nevertheless, the

immobilization process is inefficient and time-consuming due to the insensitive reactions
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between amino groups and epoxy groups [26]. Tural et al. [48] introduced epoxy groups
on the surfaces of Fe3O4@SiO2 core-shell magnetic nanoparticles by modifying with 3-
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glycidyloxypropyltrimethoxysilane and iminodiacetic acid for the covalent immobilization

of histidine-tagged recombinant benzaldehyde lyase (Figure 2). The resulting
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immobilized catalytic system was used to catalyze the kinetic resolution of racemic
acyloins, and the condensation reactions of aldehydes. Notably, the elevated yield of
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high enantiomerically pure acyloin products were achieved. The covalently immobilized
enzyme displays a considerably higher catalytic activity and thermal stability combined
with five times repeated utilization without losing its original activity.
In another study [46], the same group prepared epoxy attached MNPs and utilized as
support materials for the covalent immobilization of benzoylformate decarboxylase
under mild reaction conditions (pH 7.0, and 20 °C), as well as, harsh conditions (high
pH values, ionic strengths, etc.) (Figure 3). The enzyme loading as found to be
increased from 1.25 to 6.70 mg enzyme per gram of support in the presence of drastic

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conditions. The covalently immobilized enzyme presented good catalytic activity and
stability for the synthesis of (S)-2-hydroxypropiophenone. It showed a 53.0% enhanced
activity with reference to the activity of free counterpart. Also, the carrier-coupled
biocatalyst exhibited excellent repeated use capability and ability to maintain 95% of its
initial activity after successively reusing five times [46]. Jing et al. [43] reported the
covalent binding of carbonic anhydrase (CA) onto magnetic polymer microspheres
functionalized with an epoxy group (Figure 4). In contrast to free enzyme, the covalently

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immobilized CA displayed a better enzyme activity, elevated thermal and storage

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stability, and better reusability. It 47.6% of its original activity after successively

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recycling for six times. Fe3O4 magnetic nanoparticles fabricated by co-precipitation
strategy were coated with various ratio of SiO2 followed by functionalization with 3-

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aminopropyltriethoxysilane (APTES) and 3-mercaptopropyltrimethoxysilane (MPTMS)
(Figure 5) [42]. The functionalized nanoparticles were explored as support materials to
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attached lipase enzyme in the presence of glutaraldehyde as a cross-linking agent and
exploited their application for biodiesel production. Notably, the activity recovery and
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immobilization efficiency were gradually diminished by increasing the ratio of silica

coating on Fe3O4. However, the best activity recovery and biodiesel production yield
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were achieved by lipase attachment on Fe3O4@SiO2 support at an optimized Fe3O4: the

SiO2 ratio of 1:0.25 [42].
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Chang and Tang [60] immobilized HRP on the surface of Fe3O4@SiO2 nanoparticles
functionalized with 3-aminopropyltriethoxysilane (APTES) using glutaraldehyde as a
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cross-linker. The durability and robustness of the carrier-bound HRP were significantly
improved towards pH and temperature variations than that of the free counterpart. The
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rapid catalytic degradation capacity of 2,4-dichlorophenol by the resultant immobilized

enzyme catalyst indicates high perspective for eliminating toxic organic pollutants from
water. The Burkholderia cepacia lipase (BCL) was immobilized (via anion exchange and
covalent bonding) on a novel matrix GEAMNP developed through grafting of 2,3-
epoxypropyltrimethylammonium chloride with epoxy and quaternary ammonium group
and glutaraldehyde onto aminated magnetic nanoparticles (Figure 6) [34]. The activity
recovery of the immobilized lipase was found to be 147.4%, and the transesterification
activity was 1.5-fold greater than BCL powder. The GEAMNP immobilized lipase

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demonstrated significantly enhanced operational stability, improved reusability and

higher esters than free enzyme [34]. Of most recent, Cui et al. [30] reported an enzyme-
shielding approach to grow a protective silica layer at the surface of immobilized
enzymes on Fe3O4/silica core-shell nanospheres. The strategy involved covalent
binding of catalase enzyme onto the surface of Fe 3O4/silica core-shell nanospheres,
followed by controlled self-assembly and poly-condensation of silanes (Figure 7) [30].
Interestingly, the mesoporous organosilica layer around the conjugated enzyme

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molecules protected the enzymes from chemical, biological, and thermal denaturation

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by acting as a “shield”. Consequently, the stability and recycling capability of the

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immobilized biocatalyst was profoundly improved, and immobilized catalase preserved
70% of its initial activity after repeatedly use for nine reaction batches. Also, the strong

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covalent coupling between silica and enzymes reduced enzyme leaching and protected
from multimeric dissociation.
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3.2 Enzyme immobilization on organic polymer-modified MNPs
Generally, in-situ [62, 63] and ex-situ modification [64, 65] are two methods for
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modifying the MNPs with organic polymers. In in-situ modification mode, organic
polymers are incorporated into the precursor solution as stabilizing agents to form
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Fe3O4 nanoparticles, whereas the monomers are polymerized to synthesize polymer

coatings on the surfaces of Fe3O4 nanoparticles in ex-situ modification mode. The steric
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repulsion generated from polymer coatings weakened the magnetic and van der Waals
interactions of Fe3O4 nanoparticles that consequently prevent their aggregation and
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enhance their stability and dispersibility. For enzymes immobilization, Fe3O4

nanoparticles are commonly modified with amino polymers such as chitosan [64],
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polydopamine [65] and polyethyleneimine (PEI) [63] due to their remarkable

biodegradability and biocompatibility features. Chitosan-coated MNPs are generally
developed by the in-situ modification mode [66, 67]. Wu et al. [66] proposed a simple
and new procedure to synthesize magnetic Fe3O4-chitosan nanoparticles by in-situ
synthesis method and used as support material for the immobilization of lipase enzyme
using glutaraldehyde as cross-linker. In this process, ferrous ions were added to the
combination of chitosan and sodium tripolyphosphate solution. After adjusting the
mixture to alkaline condition, Fe(OH)2 was precipitated and oxidized to Fe3O4

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nanoparticles. The resulting Fe3O4-chitosan nanoparticles exhibited poor dispersibility

presumably due to the cross-linking between chitosan molecules or the small size of the
particles. Moreover, ex-situ modification methods namely reversed-phase suspension
[68], cross-linking [69], and embedding methods [70] have also been attempted to
prepare Fe3O4-chitosan NPs. Recently, Liu et al. [69] synthesized Fe3O4-chitosan NPs
for α-glucosidase immobilization by cross-linking and embedding method. For this,
positively charged chitosan was first coupled to negatively charged Fe3O4 followed by

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modification of chitosan on the surface of Fe3O4 by cross-linking with sodium

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tripolyphosphate. The excellent dispersibility of the resulting Fe3O4-chitosan

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nanoparticles was speculated to provide more sites for the attachment of enzymes. In
the embedding process, the Fe3O4 nanoparticles and chitosan mixture was mixed with

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NaOH solution, and the Fe3O4 nanoparticles were embedded on the chitosan
membranes pre-formed in concentrated alkaline solution. After that α-glucosidase was
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immobilized using glutaraldehyde as a coupling agent [70]. In another study, the core-
shell Fe3O4@polydopamine MNPs were fabricated by Martín et al. [65] to insolubilize
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HRP enzyme. In contrast with Fe3O4-chitosan nanoparticles, no surface

functionalization is required with any additional coupling agent (glutaraldehyde) and
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enzymes can be directly conjugated on the polydopamine film through Schiff base
reaction.
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Ren et al. [50] introduced a facile technique to immobilize lipase onto polydopamine
coated magnetic nanoparticles (PD-MNPs) (Figure 8). The method yielded a lipase
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loading capacity of 429 mg/g on PDMNPs under the optimized conditions. Immobilized
lipase presented improved pH and thermal stability profile than that of the free
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counterpart. Additionally, the PD-MNPs-insolubilized enzyme was recoverable from the

reaction medium by magnetic separation and preserved more than 70% of original
activity after reusing for 21 times. Laochai et al. [40] constructed MNPs by simple co-
precipitation method and subsequently incorporated into chitosan and poly (vinyl
alcohol) cryogel to immobilize HRP through covalent linkages resulting in MNP-CS-
PVA-HRP cryogel microbar (Figure 9). The MNP-CS-PVA- HRP cryogel microbar was
applied for the detection of hydrogen peroxide. The immobilized HRP maintained
notable enzymatic activity after reusing successively for up to 10 cycles in oxidizing the

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hydrogen peroxide. Recently, Xia et al. [38] developed PEI-modified magnetic

nanoparticles (Fe3O4–NH2–PEI NPs) by chelating with Cu2+ for the effective
immobilization of laccase by metal affinity adsorption and compared with pristine
Fe3O4–NH2 NPs. Fe3O4–NH2–PEI–Cu2+ NPs exhibited great adsorption capacity and
activity recovery of laccase (107.41% higher) than that of unmodified magnetic
nanoparticles. Immobilized laccase also showed wide-working pH and temperature
optima, and improved storage, thermal, and reusability properties. The favorable results

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indicate the high potential of Fe3O4–NH2–PEI–Cu2+ NPs for large-scale immobilization

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as well as purification of laccase simultaneously.

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Aqueous dispersible Fe3O4 nanoparticles with amine-functionalized surface were
synthesized by Sahoo et al. [37] using poly(ethylene imine) (PEI), ethanolamine (EA),

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and 2,2-(ethylenedioxy)bis(ethylamine) (EDBE) as amine precursors and used as
carrier support for immobilizing lipase (Figure 10). Among these aminated
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nanoparticles, EDBE-modified Fe3O4 nanoparticles exhibited the maximum activity for
the immobilized lipase, depicting 83.9% relative activity with reference to the free
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enzyme. Also, EDBE-Fe3O4 nanoparticles conjugated lipase revealed marked storage

and thermal stability, and easy recycling ability.
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Similarly, Candida rugosa lipase (CRL) has been covalently attached to functionalized
superparamagnetic nanoparticles (poly(GMA)-grafted Fe3O4/SiOx) synthesized by
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grafting poly-(glycidyl methacrylate) (GMA) onto the surface of Fe3O4/SiOx by atom

transfer radical polymerization [71]. The resulting immobilized lipase displayed better pH
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and thermal tolerance with a broad-ranging pH and temperature adaptability as

compared to the free counterpart. It also excellent reusability, retaining 83% of the
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original activity after reusing for six times. Lipase covalently coupled with carbodiimide
activated Fe3O4 magnetic nanoparticles presented 1.41- and 31-folds enhanced activity
and stability, respectively accompanied by marked resistance to alterations of solution
pH than that of the free enzyme [72].
3.3 Enzyme immobilization on mesoporous material-modified MNPs
Mesoporous materials are considered as promising supports for enzymes
immobilization owing to their unique characteristics of the high surface area, greater
pore volume and tunable pore size, non-toxicity, and chemical and thermal stability [73].

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Enzymes are immobilized on the surfaces or in the pores of mesoporous matrices by

physical adsorption, covalent binding or cross-linking. Different organosilanes are
commonly used to introduce amino, cyano, epoxy, or sulfhydryl groups on the surface of
mesoporous materials [74]. The presence of these functional groups create numerous
reactive sites for enzymes attachment and thereby improving the binding/loading
capacity. Additionally, the organic groups might enter into the mesoporous channel to
decrease the pore size or volume, thus preventing the leaching of enzyme molecules.

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The γ-Fe2O3 magnetic nanoparticles functionalized either with acetyl or amine groups

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were utilized to immobilize lipases by covalent binding. The resulting carrier-bound

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biocatalyst showed significantly improved operational stability and found to be stable
and reactive for 30 days [75]. Ibrahim et al. [76] covalently attached cyclodextrin

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glucanotransferase (CGTase) onto amino-functionalized magnetic double mesoporous
core-shell silica nanospheres (mag@d-SiO2 @m-SiO2-NH2), and its catalytic properties
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were evaluated. The synthesized core magnetic magnetite (Fe3O4) nanoparticles were
coated with a dense silica layer, followed by either non-functionalized or functionalized
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mesoporous silica shell, and enzyme was attached physical adsorption and covalent
binding. Notably, the covalently attached enzyme onto the activated mag@d-SiO2 @m-
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SiO2-NH2 demonstrated 98.1% immobilization yield along with a loading efficiency and
loading capacity of 96.2% and 58 µg protein [CGTase]/mg [nanoparticles]), respectively.
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In contrast with a pristine enzyme, the immobilized biocatalyst exhibited a marked

improvement in pH and thermal stability profile. Moreover, it displayed excellent
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functional stability by preserving more than 50% of original activity after recycling for 10
consecutive reaction batches.
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3.4 Enzyme immobilization on MOF-modified MNPs

Hetero-structured nanocomposites have recently received substantial attention because
of novel physicochemical properties in comparison with their single component
equivalents [77]. Among the various nanomaterials, MOFs appeared as versatile
support carriers for enzymes immobilization due to their superior properties of the large
surface area, adjustable porosity, pore size consistency, easy separation, tunable
surface, structural and functional diversity, and outstanding chemical and thermal
stability [78, 79]. The enzyme can be embedded on MOFs by three immobilization

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strategies including the surface adsorption, pore encapsulation, and co-precipitation.

Co-precipitation approach, where enzyme molecules are embedded within MOFs, can
protect enzyme molecules from chemical, thermal and biological degradation, thus
improving the stability of the immobilized enzyme [80]. The use of magnetic
nanoparticles MOF composites synthesized through combining the properties of MOFs
and Fe3O4 NPs have been reported as attractive support carriers for the immobilization
of a variety of enzymes [81, 82]. Generally, the reaction occurs between organic ligands

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and metal ions in the presence of carboxylated Fe3O4 NPs to generate these

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composites. Therefore, Fe3O4 NPs must be carboxylated yielding negatively charged

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surfaces to adsorb the positively charged metal ions and thus ensuring the growth of
MOFs on the surfaces of magnetic nanoparticles. For example, Zheng et al. [83]

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synthesized Fe3O4@ZIF-8 NPs by reacting zinc nitrate hexahydrate with 2-
methylimidazole in the presence of Fe3O4 NPs. The spherical magnetic nanoparticles
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with negative charges were generated by the solvothermal method, and Zn2+ ions were
coupled with carboxylic acid groups on Fe3O4 NPs to initiate the growth of ZIF-8 shells
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on the surfaces of these magnetic nanoparticles. The resulting magnetic-MOFs

composites were able to embed enzymes owing to the strong affinity and high loading
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capacity. In a recent study, Zhao et al. [84] developed a simple and proficient strategy to
synthesize MOF-modified magnetic nanoparticles for the elimination of methylene blue
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(MB) from simulated wastewater. The newly-engineered composites exhibit an excellent

magnetic response, large capacity and surface area, good chemical inertness and
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mechanical steadiness as well as, rapid removal of dye pollutant rendering it excellent
candidate remediation of wastewaters. Moreover, the adsorbents demonstrated
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excellent recycling capability and were usable for at least five successive reaction
cycles without losing significant activity.
Recently, a novel enzyme-shielding strategy was demonstrated by constructing the self-
assembly of supramolecular metal-organic coordination complex (tannic acid (TA) and
Fe3+) at the surface of Fe3O4/silica core-shell nanospheres embedded catalase (Figure
11) [31]. In contrast with free enzyme, the immobilized catalase exhibited improved
stability towards heat and proteolytic denaturants. The reutilizing ability of the
immobilized enzyme derivative was also substantially enhanced, and Fe3+-

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TA@Fe3O4/SiO2-catalase showed more than 50% of residual activity after 9 cycles in

comparison with a native enzyme, which preserved only 20% activity [31].
4. Economic evolution and application perspective
The worldwide market for industrially pertinent biocatalysts including those used in food,
detergent, and technical industries (such as leathers, papers, textiles, and biofuels) is
projected to grow increasingly by 2021 at an annual growth rate of 4.7% [85].
Considering the accelerating demand for naturally-derived food products, and

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sustainable energy policies requiring high shares of renewable energy sources, i.e.

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biofuels, the development of novel enzyme technologies are not only becoming an

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alternative but an implementation reality. Importantly, technical industries exhibit a great
perspective on the use of immobilized enzymes owing to bioprocess-oriented

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applications [86]. The substantial driving force for technical enzymes is renewed interest
in enzyme-mediated biofuel production utilizing lignocellulosic materials due to stringent
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environmental regulations. Therefore, enzymes involved in biofuels production
processes represent the largest portion of the technical enzyme market that is
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dominated by two major companies, Novozymes and Danisco/DuPont [86]. However,

the enzyme technologies are being inspected for integrating into paper and pulp
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industries, where catalase, xylanase, and lipase are frequently used for biomechanical
pulping, de-inking of recycled fibers, and modification of fiber properties [87, 88].
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Approximately, 35% global share of technical enzyme market is reported in Europe,

Africa, and the Middle East in 2016. Nevertheless, an annual growth rate of 6.8% and
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7.9% is expected for North America and the Asia-Pacific region, respectively through
2021 [86]. Optimizing the structure-function interactions of enzyme-assisted
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biocomposites, and trimming-down the production costs of individual support and

enzyme, as well as, the implementation costs of the overall biocatalytic process is of
profound imperious to achieve further progress in this regard (Figure 12) [85]. Coupling
or transient enzyme-assisted catalytic reactions intended to minimize community burden
related to the implementation of fossil fuels resulting in a greener, safer and
environmentally-responsive solution for the industrial establishment. The challenges
and opportunities associated with enzyme catalysis implementation will not only have to
consider revenue and marketability but integrating biomimetic approaches or “one pot”

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processes allowing for high efficiency, productivity, recyclability and stability under the
complexity of multi-chain driven specific reactions should also be considered [85].
5. Conclusions and future outlook
Immobilization is one of the most common approaches to increasing or improving the
use of enzyme catalysts for myriads of industrial applications. This technique has
already been widely pursued in biotechnology for efficiently recovering the catalyst and
product, repeatability of enzymes, and developing the native biocatalysts more robust

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and highly stable. Immobilization depends upon the choice of support material, the

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biocatalyst, and the immobilization procedure. Amid the known carrier-supports, MNPs

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are particularly alluring for enzyme immobilization perspective based on their easy
separation and recovery by the use of a magnetic field, greater surface area and high

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mass transference capacity. Numerous fascinating opportunities in enzyme catalysis
have been produced by the appropriate combination of biocatalyst with MNPs.
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However, the realization depends on their interaction necessitating the optimization
protocols to improve the catalytic activity, stability, and recycling ability of enzyme. After
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optimizing the immobilization aspects, the magnetic-enzymes bioconjugates can be

potentially employed in different fields such as detection or biosensing environmental
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contaminants, monitoring glucose and cholesterol level, manufacturing of a variety of

commercially useful pharmaceutical metabolites, and the biodegradation of hazardous
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compounds. Though several attempts have been reported, in the recent years, to
completely get the immobilization insights, but concerted efforts are still required to
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analyze the surface-function relationship, nanoparticles-enzyme binding sites and the

involvement of conformational changes in the immobilization process. From the
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intensive literature survey and several successful examples, it can be inferred that the
use of MNPs provides a very important approach for enzyme immobilization with a
definite wide perspective in enzyme catalysis and several other fields.

Acknowledgments
The literature facilities provided by Huaiyin Institute of Technology, Shanghai Jiao Tong
University, and Tecnologico de Monterrey, Campus Monterrey, Mexico are thankfully
acknowledged.

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Conflict of interest
We do not have any conflicting, competing and financial interests in any capacity.

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List of Figures

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Figure 1 A scheme for preparing co-immobilized Candida rugosa lipase and Fe3O4 NPs

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by the self-assembly of Zn2+ and AMP (Reproduced from Li et al. [35], an open-access
article distributed under the terms and conditions of the Creative Commons Attribution
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Figure 2 Preparation of Co2+-IDA-epoxy-functionalized SCMPs and their use for
immobilization of benzaldehyde lyase (Reproduced from Tural et al. [48], with
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permission from Elsevier).

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Figure 3 Schematic illustration of the preparation steps for (A) magnetic epoxy support.
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(B) Adsorption and covalent attachment of His-tagged benzoylformate decarboxylase to

magnetic epoxy support (Reproduced from Tural et al. [46], with permission from
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Figure 4 Schematic diagram of the microspheres preparation and CA immobilization
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(Reproduced from Jing et al. [43], with permission from Elsevier).

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Figure 5 Immobilization of lipase on the surface of Fe 3O4@SiO2 magnetic nanoparticles

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access article distributed under the CCBY-NC-ND license
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Figure 6 A scheme of GEAMNP (a) and GAMNP (b) synthesis for modification of
magnetic nanoparticles and Burkholderia cepacia lipase immobilization (Reproduced
from Li et al. [34], an open-access article licensed under a Creative Commons

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Figure 7 Principle of magnetic Fe3O4@silica core-shell nanosphere with silica coating

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for enzyme protection. Step 1: silanization magnetic Fe 3O4 nanoparticles; step 2:

enzyme immobilization at the surface of magnetic Fe3O4 nanoparticles by a covalent
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bond. Steps 3: silane self-assembly and 4: polycondensation (Reproduced from Cui et

al. [30], with permission from Elsevier).
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Figure 8 Two-step synthesis of lipase immobilized polydopamine coated magnetic
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Figure 9 Schematic presentation of the MNP-CS-PVA-HRP cryogel microbar process
for the detection of hydrogen peroxidase (Reproduced from Laochai et al. [40], an open

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(http://creativecommons.org/licenses/by-nc-nd/4.0/)).
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Figure 10 Preparation of amine-functionalized magnetic nanoparticles for the covalent

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conjugation of lipase (Reproduced from Sahoo et al. [37], with permission from Springer
Nature. Copyright (2016), Indian Academy of Sciences).
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Figure 11 Schematic illustration of magnetic Fe3O4@silica core-shell nanospheres with
Fe3+-TA nanocoating for enzyme protection (Reproduced from Cui et al. [31], with
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Figure 12 Representation of a multidisciplinary approach to delineate optimal
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biocatalytic processes. Implementation of biocatalysts in industrial technologies will
have to not only consider optimization of the enzyme functionality but also further, lead
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to increase in enzyme operational stability at the interface with supports used for
immobilization. (Reproduced from Chapman et al. [85], an open-access article
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Table 1 Recent Illustration of magnetic nanoparticles as versatile supporting materials for enzymes
immobilization, improved catalytic properties, and their applications.
Magnetic Enzyme Functional Immobilization Improved properties Application Reference
carrier reagent method

Fe3O4@silic Catalase TMOS, Glutaraldehyde Improved recycling potential Enzyme Cui et al.
a APTES cross-linking retaining 70% of its original shielding [30]
yolk-shell activity after 9 consecutive
nanosphere cycles
s Enhanced resistance to heat,
proteolytic agent, and
denaturants
Fe3+-TA@ Catalase APTES, Covalent Remarkably improved stability Shielding effect Cui et al.

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Fe3O4/SiO2- TMOS bonding against the proteolytic agent, to protect [31]
catalase denaturants, and heat enzymes from

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Efficient recycling ability thermal,
retaining 55% of original biological, and
activity after 9 cycles chemical
degradation

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Fe3O4@SiO Lipase APTES Covalent Good enzyme activity Enzyme Ali et al.
2@hollow attachment Thermal stability, resistance to immobilization [32]
mesoporou pH, recycling and storage
s capability
fibrous SiO2

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Fe3O4@mSi Nitrile Glutaraldeh Covalent Improved pH, thermal, Catalysis Gao et al.
O2 hydratas yde bonding mechanical and storage Production of [33]
e stability nicotinamide
GEAMNP Lipase 2,3- Anion Markedly improved operational Biodiesel Li et al.
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epoxypropyl exchange and stability, better reusability, production [34]
trimethylam covalent 96.8% fatty acid methyl esters
monium bonding conversion yield
chloride in12 h
CA-Fe3O4 Lipase Citric acid ---- Excellent long-term storage Enzyme Li et al.
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NPs stability (nearly 80% relative immobilization [35]

activity after storage for 13
days)
8-fold increased activity at pH
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10 and 1-fold at 50 °C
Magnetic Pectinex APTS Covalent Increased enzyme thermal Fruit juice Perwez et
nanoparticl 3XL bonding stability at 70 °C clarification al. [36]
e Reusability up to 5th cycle with Bioethanol
a slight decrease in enzyme production
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activity
Resistance to organic solvents
and chemical reagents
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EDBE- Lipase PEI, EA, Covalent Good thermal and storage Enzyme Sahoo et
Fe3O4 EDBE bonding stability, and easy reusability immobilization al. [37]
Fe3O4– Laccase Polyethyleni Covalent Increased enzymatic Large-scale Xia et al.
NH2–PEI mine bonding and reusability properties enzyme [38]
NPs immobilization
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HM-CSL- Lipase Ammonium Covalent Higher thermostability, storage Biocatalysis for Cui et al.
CLEAs sulfate bonding stability, and the [39]
reusability epoxidation of
Increased H2O2 tolerance oleic acid
MNP-CS- Horserad Chitosan Covalent Increased catalytic activity Detection of Laochai et
PVA-HRP ish and poly bonding Retained enzyme activity up to hydrogen al. [40]
cryogel peroxida (vinyl the 10 reuse cycles peroxide
microbar se alcohol)
Fe3O4@mSi Lipase Aniline and Covalent Wider pH‐activity and Enzyme Mahto et
O2 ammonium bonding temperature stability immobilization al. [41]
persulfate Good reusability preserving
70% of the original activity
after seven cycles
Fe3O4@SiO Lipase APTES, Glutaraldehyde 84% activity recovery Biodiesel Thangaraj
2 magnetic MPTMS cross-linking Increased catalyst stability production et al. [42]
nanoparticl
es
Magnetic Carbonic MPS Covalent Increased thermal and storage CO2 capture Jing et al.

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poly(GMA- anhydras bonding stability, and marked [43]

DVB) e reusability
microspher
es
Fe3O4@MC Porcine APTS, CPS Adsorption High stability and reusability Enzyme Shao et al.
M-41 pancreati Easy recycling and reuse immobilization [44]
c lipase
Aminosilan Esterase APETS Covalent Prolonged shelf-life No loss in Biotransformati Alex et al.
e modified bonding enzyme activity on reactions [45]
magnetic including
nanoparticl synthesis of
es ethyl acetate
and
transesterificati

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on
of vegetable oil

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production
Magnetite Benzoylf GPTMS Covalent 53% activity enhancement carboligation Tural et al.
nanoparticl ormate bonding than free enzyme reaction [46]

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es decarbox Excellent recyclability,
ylase retaining 95% of its original
activity after five repeated
cycles
Fe3O4@NH Cyclodex TEOS, Covalent Enhancement of the thermal Enzyme Ibrahim et

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2-SiO2 trin APMS bonding stability immobilization al. [47]
glycosyltr Good durability
ansferas
e
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SCMPs Benzalde GPTMS, Covalent Reusability and easy working carboligation Tural et al.
hyde IDA bonding with this support reaction [48]
lyase
Alkyl silane Lipase Trimethoxyl Hydrophobic Improved recycling ability Enzyme Wang et
modified octadecyl interaction Enhanced stability immobilization al. [49]
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magnetic silane Efficient enzyme recovery

nanoparticl
es
PD-MNPs Lipase Polydopami Covalent Enhanced pH and thermal Enzyme Ren et al.
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ne bonding stability immobilization [50]

Retained more than 70% of
initial activity after 21 repeated
cycles
BS-NSM Porcine Alkyl Adsorption Enhanced durability Hydrolysis Ponvel et
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pancreas benzenesulf and efficient recovery of olive oil al. [51]

lipase onate
PEI—poly(ethylene imine); EA—ethanolamine; EDBE—2,2-(ethylenedioxy)bis(ethylamine)
GEAMNP—2,3-epoxypropyltrimethylammonium chloride with epoxy and quaternary ammonium group
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and glutaraldehyde were grafted onto aminated magnetic nanoparticles (AMNPs)

CA-Fe3O4 NPs—Citric acid-modified magnetic iron oxide nanoparticles
APTES—3-aminopropyltriethoxysilane; MPTMS-3-mercaptopropyltrimethoxysilane
PD-MNPs— Polydopamine coated magnetic nanoparticles
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HM-CSL-CLEAs—Hybrid magnetic cross-linked lipase aggregates

TEOS—Tetraethyl orthosilicate, APMS—3-aminopropyltriethoxysilane
Fe3O4-NH2-PEI NPs–Polyethylenimine modified amine-functioned Fe3O4 nanoparticles
ABS-NSM–Alkyl benzenesulfonate nano-sized magnetite
Fe3+—TA@Fe3O4/SiO2-catalase–Hybrid organic/inorganic nanobiocatalysts; self-assembly of
supramolecular metal-organic coordination complex (tannic acid(TA) and Fe3+) at the surface of
immobilized catalase on Fe3O4/silica core-shell nanospheres to grow a protective Fe3+-TA nanocoating
SCMPs—Multifunctional Co2+ IDA epoxy functionalized silica-coated magnetic nanoparticles
GPTMS—Glycidyloxypropyltrimethoxysilane
IDA-Iminodiacetic acid
Magnetic poly(GMA-DVB) microspheres—Epoxy functionalized magnetic poly(glycidyl methacrylate-
divinyl benzene) microsphere
MPS—3-(trimethoxysilyl) propyl methacrylate

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Table 2 Methods for the synthesis of magnetic nanoparticles, their advantages, and
disadvantages. Reproduced from Xu et al. [25], an open-access article distributed under
the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
Methods Advantages Disadvantages
Physical methods
Gas-phase deposition Easy to perform Difficult to control the particle size
Electron beam lithography Well controlled inter-particle spacing Expensive and highly complex
machines requiring
Wet chemical preparation methods
Sol−gel synthesis

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Precisely controlled in size, aspect Weak bonding, low wear-resistance,
ratio, and internal high permeability

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structure
Oxidation method Uniform size and narrow size Small-sized ferrite colloids
distribution

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Chemical co-precipitation Simple and efficient Not suitable for the preparation of
highly pure, accurate stoichiometric
phase
Hydrothermal reactions Easy to control particle size and High reaction temperature,

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shapes high pressure
Flow injection synthesis Good reproducibility and high mixing Need continuous or segmented mixing
homogeneity together with a precise of reagents under a laminar flow
control of the process regime in a capillary reactor
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Electrochemical method Easy to control particle size Reproducibility
Aerosol/vapor phase High yields Extremely high temperatures
method
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Sonochemical Narrow particle size distribution Mechanism not still understood

decomposition reactions
Supercritical fluid method Efficient control of the particle size, Critical pressure and temperature
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no organic solvents involved

Synthesis using The possibility to precisely control Complex condition
nanoreactors the NP size
Microbial methods
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Microbial incubation High yield, good reproducibility, and Time-consuming

good scalability, low cost
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