Review

A Comprehensive Guide to Enzyme Immobilization: All You

Need to Know
Marina Simona Robescu * and Teodora Bavaro *

Department of Drug Sciences, University of Pavia, Viale Taramelli 12, I-27100 Pavia, Italy
* Correspondence: marinasimona.robescu@unipv.it (M.S.R.); teodora.bavaro@unipv.it (T.B.)

Abstract: Enzyme immobilization plays a critical role in enhancing the efficiency and
sustainability of biocatalysis, addressing key challenges such as limited enzyme stability,
short shelf life, and difficulties in recovery and recycling, which are pivotal for green
chemistry and industrial applications. Classical approaches, including adsorption, entrap-
ment, encapsulation, and covalent bonding, as well as advanced site-specific methods
that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These
techniques enable precise control over enzyme orientation and interaction with carriers,
optimizing catalytic activity and reusability. Key findings highlight the impact of immo-
bilization on improving enzyme performance under various operational conditions and
its role in reducing process costs through enhanced stability and recyclability. The review
presents numerous practical applications of immobilized enzymes, including their use in
the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and
in environmental biotechnology for wastewater treatment and dye degradation. Despite
the significant advantages, challenges such as activity loss due to conformational changes
and mass transfer limitations remain, necessitating tailored immobilization protocols for
specific applications. The integration of immobilization with modern biotechnological
advancements, such as site-directed mutagenesis and recombinant DNA technology, offers
a promising pathway for developing robust, efficient, and sustainable biocatalytic systems.
This comprehensive guide aims to support researchers and industries in selecting and
optimizing immobilization techniques for diverse applications in pharmaceuticals, food
processing, and fine chemicals.
Academic Editors: Yi Hu and
Gang Xu Keywords: enzyme immobilization; immobilization techniques; enzyme engineering;
Received: 23 January 2025 biocatalysis; sustainable processes
Revised: 14 February 2025
Accepted: 15 February 2025
Published: 18 February 2025

Citation: Robescu, M.S.; Bavaro, T. A 1. Introduction

Comprehensive Guide to Enzyme
In the pursuit of sustainable solutions, biocatalysis has emerged as a central strategy
Immobilization: All You Need to
Know. Molecules 2025, 30, 939. promoted by the chemical industry to align with green chemistry principles. The ability
https://doi.org/10.3390/ of biocatalysts to facilitate reactions under mild conditions while offering high selectivity
molecules30040939 positions them as a promising alternative for sustainable industrial applications [1,2].
Copyright: © 2025 by the authors. Consequently, the use of biocatalysis is expanding across various sectors, including
Licensee MDPI, Basel, Switzerland. fine chemicals, pharmaceuticals, food processing, and chemical manufacturing [3–6]. How-
This article is an open access article ever, despite their advantageous properties, the full potential of enzymes for industrial
distributed under the terms and applications remains underutilized. The challenges of enzyme stability under extreme
conditions of the Creative Commons
conditions, such as pH levels, high temperatures, and exposure to solvents, surfactants,
Attribution (CC BY) license
or metal ions, limit their broader application. Additional challenges include short shelf
(https://creativecommons.org/
licenses/by/4.0/). life and, most critically, difficulties in enzyme recovery and recycling [7–9]. To address

Molecules 2025, 30, 939 https://doi.org/10.3390/molecules30040939

Molecules 2025, 30, 939 2 of 33

these limitations, the biocatalyst can be engineered through immobilization techniques.

Immobilization has been extensively utilized (i) to improve enzyme stability and reusability,
allowing for continuous or repeated batch operations; (ii) to simplify enzyme separation
from the product, enhancing compatibility with various processes; and (iii) to reduce the
need for extensive downstream processing, making the process cost-effective, reliable,
and efficient. The engineering of enzymes through immobilization has, thus, become an
essential focus in the biocatalysis field [10]. Indeed, nowadays, enzyme immobilization has
evolved into a powerful tool for biocatalyst engineering, complementing strategies such as
recombinant DNA technology, protein engineering, high-throughput technology, genomics,
and proteomics [11,12]. The combination of recombinant DNA technology and enzyme
engineering has greatly advanced the large-scale production of enzymes with desirable
properties. Protein engineering techniques, including site-directed mutagenesis and in vitro
evolution allow for precise manipulation to achieve attributes like chemoselectivity, regios-
electivity, stereoselectivity, long-term stability, activity in high substrate concentrations, and
tolerance to organic solvents. However, these approaches are often labor-intensive, costly,
and can lack long-term operational stability. Additionally, they can pose challenges for
enzyme recovery and reuse. Despite its benefits, enzyme immobilization presents its own
challenges, including potential decreases in enzyme activity due to conformational changes
or uncontrolled orientation, risks of enzyme denaturation, altered kinetic properties, mass
transfer limitations, and reduced catalytic efficiency with insoluble substrates. However,
not all immobilization systems are affected by these issues. Robust enzyme applications of-
ten benefit from a combined approach, where protein engineering precedes immobilization,
maximizing stability and performance. To create robust biocatalysts, protein engineering
methods can be applied prior to immobilization to maximize stability and performance
across various immobilization systems. Additionally, enzyme engineering techniques can
also be applied in order to achieve a more rational immobilization protocol. By introducing
into the sequence of the enzyme specific tags or unique (un)natural amino acid residues the
orientation of the biocatalyst during immobilization can be controlled allowing to obtain a
specific interaction [13].
An effective immobilization system should securely anchor the enzyme to prevent
any unintended release that could contaminate products and lead to enzyme loss, thereby
reducing catalytic activity. Additionally, immobilization is closely linked to stability, as
only sufficiently stable biocatalysts can be reused efficiently [14,15]. However, immobiliza-
tion alone does not guarantee enzyme stabilization. In cases where poor immobilization
protocols allow uncontrolled interactions between enzyme and support, immobilization
can actually reduce enzyme stability compared to the free enzyme. Poorly designed immo-
bilization protocols, particularly those that permit uncontrolled interactions between the
enzyme and the support, can actually reduce stability compared to soluble enzymes [16].
The immobilization process, defined as the incorporation of an enzyme within or on
a porous solid support, requires careful consideration of the functional groups on both
the support and enzyme and the immobilization method itself [17]. Proper selection of
these components enables optimal immobilization outcomes. It is, however, difficult, if
not impossible, to formulate a general immobilization strategy because the method used
has to be not only protein- but also application-specific. This topic will be extensively
explored in this manuscript, providing a thorough overview of immobilization techniques
and considerations for maximizing enzyme performance.

2. Classical Non-Specific Immobilization

Classical non-specific immobilization protocols rely on the presence of reactive nat-
ural amino acids exposed on the enzyme surface that are able to react with an oppor-
 2. Classical Non‐Specific Immobilization
Molecules 2025, 30, 939 3 of 33
Classical non‐specific immobilization protocols rely on the presence of reactive
natural amino acids exposed on the enzyme surface that are able to react with an
opportunely
tunely derivatized
derivatized carrier carrier (carrier‐bound
(carrier-bound immobilization
immobilization techniques)
techniques) or with
or with other other
enzyme
enzyme molecules
molecules (carrier‐free
(carrier-free immobilization
immobilization techniques) techniques)
(Figure 1).(Figure
Classical1).non-specific
Classical non‐ im-
specific immobilization technique does not allow a fine control of the
mobilization technique does not allow a fine control of the orientation of the enzyme orientation of the
enzymeimmobilization,
during during immobilization,
even if whenevenan if enzyme
when anis enzyme is immobilized
immobilized by usingim-
by using a specific a
specific immobilization protocol, the enzyme can interact with some
mobilization protocol, the enzyme can interact with some exposed regions and not withexposed regions and
not with
others, others, suggesting
suggesting thatimmobilized
that all of the all of the immobilized proteinhave
protein molecules molecules have
a specific a specific
inaccessible
inaccessible surface area [13]. However, the immobilization of an
surface area [13]. However, the immobilization of an enzyme involving different areas ofenzyme involving
different
the enzyme areas of affect
may the enzyme may
all of its affect all (from
properties of its properties
activity to(from activity
selectivity or to selectivity
stability). Con-or
stability). Conventional enzyme immobilization techniques include
ventional enzyme immobilization techniques include both carrier-bound and carrier-free both carrier‐bound
and carrier‐free
methods (Figuremethods (Figure
1) [7,18,19]. 1) [7,18,19].these
Consequently, Consequently,
methods may these methods
involve may involve
the formation of
the formation of a strong chemical bond (covalent interaction),
a strong chemical bond (covalent interaction), weak interactions (adsorption and ionic weak interactions
(adsorption and
interactions), or ionic interactions),
including the absenceor including the absence
of any interaction of any the
between interaction
protein between
and the
the protein and the
support (cross-linking).support (cross‐linking).

Figure 1.1.Carrier-bound
Figure Carrier‐boundand carrier-free
and immobilization
carrier‐free methods.
immobilization Abbreviations:
methods. CLEs–cross-linked
Abbreviations: CLEs–cross‐
enzymes; CLECs—cross-linked enzyme crystals; CLEAs—cross-linked enzyme aggregates.
linked enzymes; CLECs—cross‐linked enzyme crystals; CLEAs—cross‐linked enzyme aggregates.
2.1. Classical Non-Covalent Immobilization
2.1. Classical Non‐Covalent Immobilization
Classical non-covalent strategies for enzyme immobilization on solid supports typi-
Classical non‐covalent strategies for enzyme immobilization on solid supports
cally rely on physical or electrostatic interactions—such as hydrophobic, van der Waals,
typically rely on physical or electrostatic interactions—such as hydrophobic, van der
hydrogen bonds, or ionic forces—between functional groups on the enzyme surface and
Waals, hydrogen bonds, or ionic forces—between functional groups on the enzyme
the support. The main drawback is the undesired release of the enzyme during the reaction
surface and the support. The main drawback is the undesired release of the enzyme
course, even when multiple weak physical interactions are used (e.g., adsorption or ion
during the reaction course, even when multiple weak physical interactions are used (e.g.,
exchange). Indeed, these approaches generally involve an overlap of several weak forces,
adsorption or ion exchange). Indeed, these approaches generally involve an overlap of
resulting in non-specific binding. However, they are the simplest and least expensive
several weak forces, resulting in non‐specific binding. However, they are the simplest and
methods, and they do not significantly alter the enzyme conformation, thereby preserving
least expensive methods, and they do not significantly alter the enzyme conformation,
its catalytic activity [20].
thereby preserving its catalytic activity [20].
Among the classical strategies for non-covalent enzyme immobilization on carriers,
Among the classical strategies for non‐covalent enzyme immobilization on carriers,
immobilization by metal affinity is also noteworthy. This approach requires recombinant
immobilization by metal affinity is also noteworthy. This approach requires recombinant
production of the protein with a six- to ten-histidine sequence (known as a His-tag) attached
production of the protein with a six‐ to ten‐histidine sequence (known as a His‐tag)
to its N- or C-terminus. In this review, this method is further discussed in Section 3.1.1.

2.1.1. Entrapment
Entrapment is an immobilization technique that involves enclosing enzymes or cells
within a fiber network. It can also be described as the encapsulation of enzymes within a
Molecules 2025, 30, 939 4 of 33

support material, either with a lattice structure or integrated into polymer membranes. By
carefully adjusting the pore size of the polymeric network, it is possible to prevent enzyme
leakage while allowing the free diffusion of substrates and products [21].
Isolated enzymes have been immobilized using this technique. Alkaline protease
was entrapped in mesoporous silica and zeolite (immobilization yield 63.5% and 79.77%,
respectively) and utilized as a milk coagulant in the production of dairy products, while
laccase was immobilized on alginate beads for dye removal from water [21–23].
One of the main advantages of this technique is that enzymes do not chemically interact
with the polymers, which significantly reduces the risk of denaturation. Additionally,
entrapment enables high enzyme loading capacity, enhances mechanical stability, and is
relatively inexpensive to implement. The material used for entrapment can also be tailored
to optimize the enzyme microenvironment by adjusting properties such as pH, polarity, or
amphiphilicity. Despite its benefits, entrapment has certain limitations. As polymerization
progresses, the resulting increase in matrix thickness can lead to mass transfer resistance,
making it difficult for substrates to reach the enzyme active site. Furthermore, if the pore
sizes of the support material are too large, enzymes may leak out. The most widely used
method for enzyme entrapment is the gelation of polycationic or polyanionic polymers
using multivalent counterions. Other techniques include photopolymerization, sol-gel
processes, and electropolymerization [24].
A more recent advancement in enzyme immobilization is the use of membranes as
carriers. Enzyme immobilization on membranes (EIM) has emerged as a promising strategy
for enhancing enzymatic stability, reusability, and efficiency in biocatalytic applications [25].
In this context, fumarase has been entrapped within polysulfone membranes for the pro-
duction of L-malic acid, a compound widely used in food and pharmaceutical industries.
By confining the enzyme within the membrane’s porous network, this system maintains
high enzymatic activity over extended periods, though substrate diffusion may become a
limiting factor. Another example is the co-immobilization of glucose oxidase and catalase
in polymeric composite membranes, where both enzymes work synergistically for glucose
oxidation. This multienzymatic system is particularly valuable in biosensing applications,
ensuring selective enzymatic reactions while reducing external interferences [26].

2.1.2. Encapsulation
Encapsulation is similar to entrapment in that both enzymes and cells are suspended
in solutions but within a controlled environment. The encapsulation technique is specifi-
cally intended for sensitive enzymes and cells, confining them within small vesicles that
have porous membranes [27]. Ionotropic gelation of alginates and silica-based nanoporous
sol-gel glasses has proven to be an effective method for enzyme encapsulation. Few
examples are reported in the literature regarding enzyme immobilization through encap-
sulation. Notably, Nitto Chemical (now Mitsubishi Rayon, Tokyo, Japan) developed a
process in which the conversion of acrylonitrile to acrylamide, catalyzed by bacterial nitrile
hydratases, is achieved. This biotransformation is performed using immobilized whole
cells encapsulated within a cross-linked gel composed of 10% (w/v) polyacrylamide and
dimethylaminoethylmethacrylate [18].
Recently, horseradish peroxidase encapsulated into tyramine-alginate beads was
applied for phenol degradation in the treatment of wastewater. After four cycles, the
immobilized horseradish peroxidase preserved >60% activity, with a phenol removal
efficiency reported at 96% [28]. α-Lactalbumin nanotubes were used as carriers for lipase
immobilization. This derivative released 50% more free fatty acids compared to soluble
lipases. In low-fat cheeses, these nanotubes doubled the release of free fatty acids compared
to regular cheeses with the same fat content, enhancing the flavor of the low-fat cheese [29].
Molecules 2025, 30, 939 5 of 33

However, this technique comes with certain constrains. One of the main issues is the
diffusion problem, which can be particularly severe and may result in membrane rupture if
the reaction products accumulate too quickly [30].

2.1.3. Adsorption
Immobilization by adsorption is the easiest and least invasive reversible immobi-
lization technique. This method provides weak enzyme binding (van der Waals and
hydrophobic interactions), and lipases immobilized on hydrophobic resins, especially
polymethyl methacrylates, are extensively used. One of the most notable examples of
lipase adsorption onto organic resins is the widely utilized Candida antarctica lipase B
(CaLB), commercially available in its immobilized form as Novozym 435® . This derivative
features the enzyme adsorbed onto a macroporous resin composed of polymethyl/butyl
methacrylate cross-linked with divinylbenzene, and the immobilized lipase activity was
preserved after 3 h at 60 ◦ C [31]. Hydrophobic resin-immobilized lipases, including CaLB,
are commonly used for the production of various edible oils, such as cocoa butter sub-
stitutes, fats for infant formulas, emulsifiers, and omega-3 fish oil derivatives [32,33].
In addition, the use of immobilized lipases as chemo- and regioselective catalysts is es-
pecially valuable in organic chemistry for the synthesis of modified carbohydrates for
pharmaceutical applications [34–38].
Lipases typically have a hydrophobic surface with a lid that shields the active site,
likely to prevent the hydrolysis of non-lipid esters within the cell. The enzyme is activated
when the lid opens, which occurs in hydrophobic environments such as the surface of a
lipid droplet. By using a hydrophobic carrier, the lipase can bind while also keeping the lid
locked in its open position.
Other examples of enzymes immobilized by adsorption and applied in the pharmaceu-
tical field involve transaminases, which are often used for the synthesis of chiral molecules.
These enzymes, immobilized on hydrophobic resins, enable stereoselective reactions, which
are essential for the production of pharmaceutical active ingredients. For instance, im-
mobilized transaminases have been used in the synthesis of drugs for the treatment of
diabetes, such as sitagliptin, or other compounds with high enantiomeric purity required
in therapeutic applications [16,39].
Cellulases and laccases immobilized by adsorption are widely used in the textile indus-
try [21]. Recently, laccase from Trametes versicolor was adsorbed on TiO2 −ZrO2 −SiO2 with
nearly quantitative immobilization yields, and it was successfully used in the degradation
of dyes from textiles [40]. This immobilization technique has been also used to adsorb the
laccase onto polyvinylidene fluoride membranes to degrade pharmaceutical pollutants in
wastewater treatment. This method allows for easy enzyme recovery and reuse, although
desorption remains a challenge. Similarly, lipase has been immobilized on polypropylene
membranes for biodiesel production, where it catalyzes the transesterification of oils [26].
Immobilization through simple adsorption or ionic binding (as described in the fol-
lowing paragraph) has the drawback of potential enzyme leaching in aqueous media,
depending on the pH and ionic strength. This limitation restricts its use to water-free
systems. To address this issue, a hybrid tailor-made immobilization method has been
developed for membrane enzymes. Specifically, equine kidney γ-glutamyl-transpeptidase
(ekGGT), a membrane enzyme used for the synthesis of γ-glutamyl amino acids, has been
immobilized on a heterofunctional carrier with a high immobilization yield and activity
recovery (93% and 88%, respectively). This carrier combines hydrophobic alkyl chains
and aldehyde groups, enabling concurrent adsorption interactions that mimic the lipid
environment of cellular membranes and covalent immobilization to stabilize the enzyme,
thereby preventing potential leaching [41].
Molecules 2025, 30, 939 6 of 33

2.1.4. Ionic Binding

Enzymes can be immobilized through ionic binding using an ion-exchange resin. The
selection of a cationic or anionic resin is dictated by the enzyme’s net surface charge, which
depends on its isoelectric point and the pH of the immobilization solution. The first large-
scale industrial application of an immobilized enzyme was reported in 1969 by Tanabe
Seiyaku, involving the use of an aminoacylase from Aspergillus oryzae immobilized by ad-
sorption on DEAE-Sephadex (cross-linked dextran functionalized with diethylaminoethyl
groups) for the synthesis of an L-amino acid [42].
As previously described, ionic immobilization presents similar drawbacks to ad-
sorption immobilization. In this case as well, hybrid immobilization methods, combin-
ing ionic immobilization followed by covalent immobilization, have been developed for
multimeric enzymes. For example, nucleoside phosphorylases from B. subtilis were im-
mobilized onto Sepabeads coated with poly (ethyleneimine) (Sep-PEI) by ionic binding
and, finally, the derivative was treated with polyaldehyde macromolecules (20% oxidized
dextran) that, reacting both with the free amino groups of the enzyme and PEI, afforded
a covalent multipoint cross-linking between the protein subunits and the support (76%
immobilization yield) [43].

2.2. Classical Covalent Immobilization

Covalent immobilization requires the presence of two mutually reactive chemical
groups on the enzyme and on a carrier surface (carrier-bond) or another protein molecule
(carrier-free). Classical covalent immobilization strategies exploit the reactivity of endoge-
nous functional groups present in the side chains of the amino acids of the enzyme. Table 1
shows some of the reactive groups present in naturally occurring amino acids that are used
for covalent immobilization. Amines and thiols are both good nucleophiles and are the
Molecules 2025, 30, x FOR PEER REVIEW
most
Molecules 2025, 30, x FOR PEER widely exploited residues for covalent immobilization. Carboxylic acid groups have 77 of
REVIEW of 35
35
Molecules 2025, 30, x FOR PEER REVIEW 7 of 35
to be activated to make them reactive toward nucleophiles [13].

Most common
Table 1. Table Mostreactive
1. Most commongroups naturally
reactive presentnaturally
groups on enzyme surfaceon
present usedenzyme
for immobilization
surface used
used for
for
Table 1. common reactive groups naturally present on enzyme surface
throughTable 1. bond
covalent Mostformation.
immobilization
common reactive groups naturally present on enzyme surface used for
through covalent bond formation.
immobilization through covalent bond formation.
immobilization through covalent bond formation.
Reactive GroupGroup
Reactive Structure Amino
Amino Acid
acid
Reactive Group Structure Amino acid
Reactive Group Structure N‐terminus Amino acid
N-terminus
N‐terminus
Primary
Primary amine
amineamine
Primary N‐terminus
Primary amine ‐amino groups
ε-amino
‐amino groupsgroups
of of Lysine
of Lysine
Lysine
‐amino groups of Lysine
C‐terminus
C-terminus
C‐terminus
Carboxylic acid C‐terminus
Glutamic acid acid
Carboxylic acid acid
Carboxylic Glutamic
Glutamic acid
Carboxylic acid Glutamic acid acid
Aspartic
Aspartic acid
Aspartic acid
Aspartic acid
Thiol
Thiol Thiol Cysteine
Cysteine
Cysteine
Thiol Cysteine
Imidazole
Imidazole Histidine
Histidine
Imidazole
Imidazole Histidine
Histidine

Carrier‐Bound
Carrier‐Bound
Carrier‐Bound
AmongUnlike the classical
Unlike methods of
immobilization
immobilization viacovalent
via enzymecovalent
cross‐linking,
cross‐linking, immobilization,
covalent bondingthere
bonding are also
involves
involves attaching
attaching
strategies that
enzymes Unlike
do not
to immobilization
require
support any
materials, via
supportsuch cross‐linking,
(CLEs,
as CLEAs,
porous covalent
and
silica, CLECs, bonding
Figure
polyacrylamide, involves
1). This
agarose, attaching
topic
or porous
enzymes to support materials, such as porous silica, polyacrylamide, agarose, or porous
enzymes
has been extensively
glass, to support
forming described materials,
and such
reviewed as porous
by Roger silica, polyacrylamide,
Sheldonleakage
[16,44] and agarose,
will or porous
notattachment
be
glass, forming aa robust
robust andand stable
stable connection,
connection, minimizing
minimizing leakage issues.
issues. The
The attachment
glass,
addressed
of in
the forming
this review.
enzyme a robust
toIt is
the and
worth stable
support connection,
highlighting
can occur that
byminimizing
cross-linking
establishing leakage
has issues.
been The
adopted
multi‐point or attachment
as a
single‐point
of the enzyme to the support can occur by establishing multi‐point or single‐point
of the
recent application enzyme
covalent bonds for EIM.
bonds to the support
One key
between theexamplecan
functional occur
is the by establishing
immobilization
groups on the multi‐point or
of β-galactosidase
the enzyme’s
enzyme’s surface and single‐point
andonsuitable
suitable
covalent between the functional groups on surface
covalent
polyvinylidene
reactive bonds on
fluoride
groups between
membranes
the thesupport
solid functional
using groups
glutaraldehyde
(Table 2). on
(GA)
Covalentthecross-linking.
enzyme’s surface
immobilization This and suitable
enzyme
mediated by the
the
reactive groups on the solid support (Table 2). Covalent immobilization mediated by
reactive
derivative
carrier groups
is used
involvesin the on the solid
production
modifying support
the of (Table 2). Covalent
galacto-oligosaccharides
polymer structure through immobilization
(GOS), activation
through support
support mediated
which serve
activation as by
achieved the
by
carrier involves modifying the polymer structure achieved by
carrier
prebiotic
the involves
ingredients
addition in
of modifying
functional
reactive the
foods.
moleculespolymer
The structure through
cross-linking
[18,45]. processsupport activation
significantly achieved by
improves
the addition of reactive molecules [18,45].
the addition
Covalent of immobilization
reactive molecules by [18,45].
epoxy carriers
carriers involves
involves the
the reaction
reaction of of free
free amino
amino
Covalent immobilization by epoxy
groups, Covalent
such as immobilization
lysine residues, by
on epoxy
the carriers
surface of involves Athe
enzymes. reaction of free
straightforward amino
method to
groups, such as lysine residues, on the surface of enzymes. A straightforward method to
groups,
achieve this such
this is as
is by lysine
by exploitingresidues,
exploiting the on
the epoxy the surface
epoxy groups
groups of of enzymes.
of functionalizedA straightforward
functionalized resins
resins (e.g., method
(e.g., methacrylate
methacrylate to
achieve
achieve
carriers), this
whichis byundergo
exploiting the epoxy groups
nucleophilic attack atof mild
functionalized
pH values resins
(pH (e.g.,
8) by methacrylate
the available
available
carriers), which undergo nucleophilic attack at mild pH values (pH 8) by the
carriers), which undergo nucleophilic attack at mild pH values
amino groups on the enzyme’s surface. This reaction leads to the formation of stable (pH 8) by the available
Molecules 2025, 30, 939 7 of 33

enzyme retention, although excessive reticulation may slightly reduce enzymatic efficiency
due to steric hindrance. A similar strategy has been employed for lipase immobilized on
alumina hollow fiber membranes, facilitating hydrolysis reactions in industrial settings.
The high surface area and strong cross-linking bonds ensure prolonged activity, making
this an attractive option for large-scale enzymatic processes [26].

Carrier-Bound
Unlike immobilization via cross-linking, covalent bonding involves attaching enzymes
to support materials, such as porous silica, polyacrylamide, agarose, or porous glass,
forming a robust and stable connection, minimizing leakage issues. The attachment of
the enzyme to the support can occur by establishing multi-point or single-point covalent
bonds between the functional groups on the enzyme’s surface and suitable reactive groups
on the solid support (Table 2). Covalent immobilization mediated by the carrier involves
modifying the polymer structure through support activation achieved by the addition
Molecules 2025, 30, x FOR PEER REVIEW 8 of of
35
Molecules 2025, 30, x FOR PEER REVIEW 8 of 35
Molecules 2025, 30, x FOR PEER REVIEW
reactive molecules [18,45]. 8 of 35

Table 2. Most common functional groups present on carriers and used for covalent bonds
Table 2. Most common functional groups present on carriers and used for covalent bonds with
with
Tableenzymes.
2. Most common functional groups present on carriers and used for covalent bonds with
Table 2. Most common functional groups present on carriers and used for covalent bonds with
enzymes.
enzymes.
enzymes.
Functional Reactive Group
Functional Structure Binding Reactive Group
Group
Functional Structure Binding of the Enzyme
Reactive Group
Functional
Group Structure Binding Reactive Group
of the Enzyme
Group Structure Binding
Nucleophilicattach of the Enzyme
attachand Nucleophilic
Group Nucleophilic of thegroups
Nucleophilic Enzyme
groups(‐NH2
Epoxy and epoxy ring
Nucleophilic attach and Nucleophilic groups (‐NH2
Epoxy Nucleophilic attach and Nucleophilic
epoxy ring opening and
(-NH‐SH)2 and groups
-SH) (‐NH 2
Epoxy opening
epoxy ring opening and ‐SH)
epoxy ring with
Activation openinga and ‐SH)
Activation
Activationwith
with aa Primary
Primary amines
amines(terminal‐
Amino Activation
dialdehyde; with a base Primary
Schiff amines (terminal‐
Amino dialdehyde;
dialdehyde; Schiff
Schiffbase
base Primary
NH 2 andamines
(terminal-NH (terminal‐
Lys side chains)
2 and Lys
Amino dialdehyde;
formation Schiff base NH2 and Lys side chains)
formation
formation NHside
2 and Lys
chains) side chains)
formation
Activation with a
Activation
Activationwith
with aa Primary
Primary amines
amines(terminal‐
Activation
dialdehyde; with a base Primary
Schiff amines (terminal‐
dialdehyde;
dialdehyde;Schiff
Schiffbase Primary
NH 2 and amines
Lys
base (terminal-NH2 and (terminal‐
side chains)
Lys
dialdehyde;
formation Schiff base NH2 and Lys side chains)
formation
formation NH 2 and
side Lys side chains)
chains)
formation Imidazole, thiol and primary
Activation with DVS; Imidazole,
Imidazole, thiol and
thiol primary
and
Activation
Activation with
with DVS;
DVS; Imidazole,
amines thiol
(dependingand primary
on the
Activation with
C‐X bond formation DVS; amines
primary(depending
amines on the
C‐X bond
C-Xbond formation
bondformation
formation amines
pH) (depending on the
C‐X pH)
(depending on the pH)
Activation with CnBr; pH)
Activation
Activationwith
withCnBr; Primary
CnBr; Primary
Primary amines
amines(terminal‐
Activation with
imidocarbonate CnBr;
bond amines (terminal‐
imidocarbonate
imidocarbonatebond bond Primary
NH amines
2 at mild
(terminal-NH pH)(terminal‐
at mild
imidocarbonate
formation bond NH2 at mild pH)2
formation
formation NH 2 at mild pH)
pH)
Hydroxy formation
Activation with CDI;
Hydroxy
Hydroxy Activation
Activationwith
withCDI; Primary
CDI; Primary
Primary amines
amines(terminal‐
Hydroxy Activation
carbamate with
bond CDI; amines (terminal‐
carbamate
carbamate bond
bond Primary
NH 2 andamines
(terminal-NH (terminal‐
Lys side chains)
2 and Lys
carbamate
formation bond NH2 and Lys side chains)
formation
formation NHside
2 and Lys
chains) side chains)
formation
Activation with epoxy
Activation
Activation with
withepoxy
epoxy Nucleophilic groups (‐NH2
Activation
groups with
(GLYMO); epoxy
groups
groups(GLYMO);
(GLYMO); Nucleophilic groups (‐NH2
groups (GLYMO);
Nucleophilic attach andNucleophilic
and ‐SH)
Nucleophilic groups
groups(‐NH2
Nucleophilicattach
Nucleophilic attachand and ‐SH)
Nucleophilic attach and and
epoxy ring opening (-NH
‐SH) 2 and -SH)
and epoxy
epoxy ring
ring opening
epoxy ring
Activation opening
with
opening with thiolthiol
Activation
Activation with
groups; thiol thiol
exchange ‐SH
Activation
groups; thiolwith thiol ‐SH
exchange
groups; thiol exchange ‐SH
(S‐S bond)
groups;
(S‐S bond)thiol exchange -SH
(S‐S
Legend: DVS = divinyl sulfone; CNBr bond)
(S-S bond) bromide; CDI = N,N’‐carbonyldiimidazole;
= cyanogen
Legend: DVS = divinyl sulfone; CNBr = cyanogen bromide; CDI = N,N’‐carbonyldiimidazole;
Legend:
GLYMODVS
Legend: DVS = divinyl sulfone;
CNBrCNBr = cyanogen
bromide;bromide;
= (3‐glycidyloxypropyl)trimethoxysilane.
= divinyl sulfone; = cyanogen CDI = N,NCDI = N,N’‐carbonyldiimidazole;
′ -carbonyldiimidazole; GLYMO = (3-
GLYMO = (3‐glycidyloxypropyl)trimethoxysilane.
glycidyloxypropyl)trimethoxysilane.
GLYMO = (3‐glycidyloxypropyl)trimethoxysilane.

Covalent immobilization by epoxy carriers involves the reaction of free amino groups,
such as lysine residues, on the surface of enzymes. A straightforward method to achieve
this is by exploiting the epoxy groups of functionalized resins (e.g., methacrylate carriers),

Scheme 1. Covalent immobilization of enzymes to epoxy methacrylate resins.

Scheme 1. Covalent immobilization of enzymes to epoxy methacrylate resins.
 NH2 at mild pH)
formation
Hydroxy Activation with CDI;
Primary amines (terminal‐
Molecules 2025, 30, 939 carbamate bond
NH2 and Lys side chains)8 of 33
formation
Activation with epoxy
which undergo nucleophilic attack atgroups
mild pH(GLYMO);
values (pH 8) Nucleophilic groups
by the available (‐NH
amino groups
2

Nucleophilic
on the enzyme’s surface. This reaction attach
leads to the and andof‐SH)
formation stable secondary amine
epoxy ring opening
bonds, effectively immobilizing the enzyme (Scheme 1). Oxirane groups are also able to
Activation
react with various nucleophilic groups, withto
in addition thiol
primary amino groups, such as thiol
groups; thiol exchange
groups, forming strong thioether bonds. However, the reactivity‐SH of epoxy groups is low
(S‐S bond)
and, generally, a long incubation time is required for immobilization (at least 24 h). First,
Legend:
the DVShas
enzyme = divinyl
to comesulfone; CNBr
in close = cyanogen
contact with thebromide; CDI = N,N’‐carbonyldiimidazole;
carrier (adsorption) and, subsequently,
GLYMO
the = (3‐glycidyloxypropyl)trimethoxysilane.
covalent bond is formed.

Scheme 1. Covalent
Scheme 1. Covalent immobilization
immobilization of
of enzymes
enzymes to
to epoxy
epoxy methacrylate
methacrylate resins.
resins.

The amino groups

The amino groupson onthe
thefunctionalized
functionalized carrier
carrier cancan react
react with with
the the enzyme
enzyme afterafter
pre‐
pre-activation
activation withwith glutaraldehyde
glutaraldehyde (GA)(GA)[46] [46] or other
or other safersafer bifunctional
bifunctional reagents
reagents (e.g.,(e.g.,
2,5‐
2,5-diformylfuran [47]) (Scheme 2). This process involves the interaction
diformylfuran [47]) (Scheme 2). This process involves the interaction of the resulting of the resulting
aldehyde
aldehyde groups
groups with
with the
the enzyme’s
enzyme’s aminoamino groups,
groups, generally
generally atat basic
basic pH pH values
values (pH(pH 10),
10),
leading
Molecules 2025, 30, x FOR PEER REVIEW to the rapid formation of Schiff bases, which are unstable under
leading to the rapid formation of Schiff bases, which are unstable under acidic conditions. acidic conditions.
9 of 35
These
Theseimines
iminescan subsequently
can subsequently be reduced (e.g.,(e.g.,
be reduced usually by NaBH
usually , NaBH
by 4NaBH CN, or 2-picoline
4, 3NaBH3CN, or 2‐
borane
picolinecomplex), resulting resulting
borane complex), in irreversible enzyme immobilization,
in irreversible although this
enzyme immobilization, may carry
although this
the risk
may Many of diminishing
carry examples
the risk of the activity
of diminishing of
biotransformations the biocatalyst
catalyzed
the activity of the[16,18,45,48].
by biocatalyst If 2,5-diformylfuran
enzymes immobilized
[16,18,45,48]. on If
epoxy
2,5‐
(DFF)
diformylfuran (DFF) is used, the imine bond is also stable at acidic pH values thanks of
and is used,
amino the imine
carriers bond
have is
been also stable
reported at
foracidic
the pH values
synthesis thanks
of to the
compounds conjugation
relevant to
to
the formed
pharmaceutical
the imine
conjugation of bond
and with
thefood the
fields
formed furan ring, thus avoiding the reduction step
[21].bond with the furan ring, thus avoiding the reduction
imine [47].
step [47].

Scheme 2. Immobilization of enzymes on amino functionalized carrier. (A) Derivatization with GA;
Scheme 2. Immobilization of enzymes on amino functionalized carrier. (A) Derivatization with GA;
(B) derivatization with DFF.
(B) derivatization with DFF.

Many examples of biotransformations catalyzed by enzymes immobilized on epoxy

The hydroxyl groups present in polysaccharide‐based carriers are very versatile and
and amino carriers have been reported for the synthesis of compounds relevant to pharma-
can be derivatized by a plethora of activations, allowing the covalent attachment of
ceutical and food fields [21].
enzymes by different binding chemistries. Agarose is one of the most inert polymers that
is widely used for enzyme immobilization after activation of hydroxyl groups (Scheme 3)
[20,49–54].
Molecules 2025, 30, 939 9 of 33

The hydroxyl groups present in polysaccharide-based carriers are very versatile and
can be derivatized by a plethora of activations, allowing the covalent attachment of enzymes
by different binding chemistries. Agarose is one of the most inert polymers that is 10
Molecules 2025, 30, x FOR PEER REVIEW widely
of 35
used for enzyme immobilization after activation of hydroxyl groups (Scheme 3) [20,49–54].

Scheme 3. Chemistry of functionalized agarose. Legend: APTES = (3‐aminopropyl)triethoxysilane;

Scheme 3. Chemistry of functionalized agarose. Legend: APTES = (3-aminopropyl)triethoxysilane;
CDI = N,N’‐carbonyldiimidazole; GLYMO = (3‐glycidyloxypropyl)trimethoxysilane; CNBr =
CDI = N,N′ -carbonyldiimidazole; GLYMO = (3-glycidyloxypropyl)trimethoxysilane; CNBr = cyanogen
cyanogenDTNB
bromide; bromide;
= 2,2DTNB = 2,2′‐dinitro‐5,5′‐thiobenzoic
′ -dinitro-5,5 acid; DVS
′ -thiobenzoic acid; DVS = divinyl = divinyl
sulfone; ECH =sulfone; ECH =
epichlorhydrin;
epichlorhydrin; GA = glutaraldehyde;
GA = glutaraldehyde; OcA = octylamine. OcA = octylamine.

Glyoxyl‐agarose, glutaraldehyde
Glyoxyl-agarose, glutaraldehyde glyoxyl‐agarose,
glyoxyl-agarose, and APTES‐agarose,
and APTES-agarose, with
with different
different
spacer spacerreact
groups, groups,
with react with theusing
the enzymes enzymes using
the same the same
binding binding
chemistry chemistry
described in
described
Scheme 2. in
In Scheme 2. In
fact, these fact, thesecarriers
derivatized derivatized carriers
contain contain free
free aldehyde aldehyde
groups, groups,
enabling the
enabling the multipoint covalent attachment of enzymes through Schiff base formation
with the free amino groups of lysine residues. By varying the length of the spacer groups,
the properties of the final enzyme derivative can be modulated since longer spacer arms
allow an immobilized biocatalyst to be obtained with more flexibility as well as more
distance from the carrier surface. Depending on the enzyme and the application, these
 Molecules 2025, 30, 939 10 of 33

multipoint covalent attachment of enzymes through Schiff base formation with the free 11 of 3
Molecules 2025, 30, x FOR PEER REVIEW
amino groups of lysine residues. By varying the length of the spacer groups, the proper-
ties of the final enzyme derivative can be modulated since longer spacer arms allow an
immobilized biocatalyst to be obtained with more flexibility as well as more distance from
Aldehyde‐agarose‐based
the carrier surface. Depending carriers
on theare likely and
enzyme the most commonlythese
the application, usedproperties
supportscanfor protein
immobilization.
negatively Moreover,
or positively influence the
theenzymes
outcome of immobilized on glyoxyl‐agarose
the final derivative. Aldehyde-agarose- have been
successfully
based carriersapplied
are likelytothe
biotransformations
most commonly used under flow conditions
supports for protein [55–58].
immobilization.
Moreover, the enzymes immobilized on glyoxyl-agarose have been
As previously reported, the octyl‐glyoxyl‐agarose carrier has been successfully applied to
successfully
biotransformations
employed in the hybrid,under flow conditionsimmobilization
tailor‐made [55–58]. of membrane proteins [41].
As previously reported, the octyl-glyoxyl-agarose
The immobilization using GLYMO‐agarose also occurs, carrier has been successfully
as described em-
in Scheme 1.
ployed in the hybrid, tailor-made immobilization of membrane proteins [41].
Hydroxyl group activation with divinyl sulfone (DVS) can react mainly with
The immobilization using GLYMO-agarose also occurs, as described in Scheme 1.
imidazole and thiol groups of the amino acid side chain, but some reactivity with amino
Hydroxyl group activation with divinyl sulfone (DVS) can react mainly with imida-
and phenol groups has also been observed depending on the pH [59]. DVS groups are
zole and thiol groups of the amino acid side chain, but some reactivity with amino and
much groups
phenol more reactive compared
has also been observedtodepending
epoxy groups,
on the pH since
[59]. they are able
DVS groups to covalently
are much
immobilize
more reactiveenzymes
compared without
to epoxy requiring previous
groups, since they adsorption of the enzyme
are able to covalently to the surface
immobilize
Moreover, DVS supports can be used in a wide range of pH values
enzymes without requiring previous adsorption of the enzyme to the surface. Moreover, compared to glyoxyl
agarose,
DVS which
supports canisbegenerally
used in aemployed
wide rangeatofalkaline
pH valuespHcompared
values. Moreover, compared to an
to glyoxyl-agarose,
which is generally employed
aldehyde‐based at alkaline pH values.
carrier, DVS‐activation allowsMoreover,
a stablecompared to an aldehyde-
enzyme–carrier linkage to be
based carrier, DVS-activation allows a stable enzyme–carrier linkage to be directly
directly obtained without the need for the reduction step (Scheme 4). Different enzymes obtained
without
such as the need for the P1‐A04
ketoreductase reduction stepCodexis
from (Scheme [60],
4). Different
as well enzymes,
as lysine such as ketore-
cyclodeaminase from
ductase P1-A04 from Codexis [60], as well as lysine cyclodeaminase from Streptomyces
Streptomyces pristinaespiralis [61], were immobilized at mild pH on DVS‐agarose through
pristinaespiralis [61], were immobilized at mild pH on DVS-agarose through interaction
interaction with His residues obtaining good immobilization yields but unfortunately low
with His residues obtaining good immobilization yields but unfortunately low recov-
recovered
ered activity.activity.
This was This was attributed
attributed to a strongtorigidification
a strong rigidification
of the protein ofstructures
the protein structure
after
after covalent
covalent immobilization.
immobilization.

Scheme4.4.Immobilization
Scheme Immobilizationof enzymes on DVS
of enzymes functionalized
on DVS carrier.carrier.
functionalized

The hydroxyl groups of the agarose (vicinal diols) can react with cyanogen bromide
The hydroxyl groups of the agarose (vicinal diols) can react with cyanogen bromide
(CNBr) to give the reactive cyclic imido-carbonate [62]. This primarily reacts with the
(CNBr) to give the reactive cyclic imido‐carbonate [62]. This primarily reacts with the N
N-terminal amino acid under mildly basic conditions, allowing for single-point immobi-
terminal
lization amino
(Scheme acid under mildly
5). CNBr-activated basic
agarose has conditions,
proven allowing
to be an effective forforsingle‐poin
carrier the
immobilization (Scheme 5). CNBr‐activated agarose has proven to be an effective
immobilization of native human epidermal growth factor (hEGF), ensuring proper orienta- carrie
for the
tion immobilization
of the of native
protein to prepare human
an active epidermal
and flexible growth
supported factor
EGF (hEGF),
for tissue ensuring prope
engineering.
orientation
The of the
bioconjugate wasprotein
obtainedtoin prepare
65% yieldan active
after and flexible supported EGF for tissue
3 h [63].
Different immobilization binding chemistry can be
engineering. The bioconjugate was obtained in 65% yield used N,N′ -carbonyldiimidazole
when after 3 h [63].
(CDI) is employed to activate the agarose. CDI is a highly reactive carboxylating agent that
contains two acylimidazole leaving groups, which form reactive carbonyl groups on the
hydroxyl support. This conjugate reagent has been successfully used in peptide synthesis
and also in the immobilization of enzyme and affinity ligands in a chromatography matrix.
The activation process of the carrier has to be performed in anhydrous media due to the sus-
ceptibility to hydrolysis of CDI [50]. Immobilization on agarose-CDI is another technique

Scheme 5. Covalent immobilization of enzymes to CNBr‐agarose.

 Scheme 4. Immobilization of enzymes on DVS functionalized carrier.

The hydroxyl groups of the agarose (vicinal diols) can react with cyanogen
Molecules 2025, 30, 939 11 of 33
(CNBr) to give the reactive cyclic imido‐carbonate [62]. This primarily reacts wit
terminal amino acid under mildly basic conditions, allowing for sing
that enables multipoint
immobilization enzyme-support
(Scheme interaction through
5). CNBr‐activated agarose thehas
formation
proven of to
carbamate
be an effectiv
Molecules 2025, 30, x FOR PEER REVIEW
bonds between the surface lysins of the enzyme and the activated support (Scheme 6).
for the immobilization of native human epidermal growth factor (hEGF), ensuring
Few examples of enzyme immobilization via CDI-carrier are currently reported in the
orientation of the protein to prepare an active and flexible supported EGF fo
literature [64]. A very interesting example involves the conjugation of ovalbumin, used as
aengineering.
model antigen,The
ontobioconjugate was obtained
magnetite nanoparticles in 65%asyield
for application after 3vaccines
particulate h [63].[50].
used in peptide synthesis and also in the immobilization of enzyme and affinity
in a chromatography matrix. The activation process of the carrier has to be perfo
anhydrous media due to the susceptibility to hydrolysis of CDI [50]. Immobiliza
agarose‐CDI is another technique that enables multipoint enzyme‐support int
through the formation of carbamate bonds between the surface lysins of the enzy
the activated support (Scheme 6). Few examples of enzyme immobilization v
carrier are currently reported in the literature [64]. A very interesting example i
the conjugation of ovalbumin, used as a model antigen, onto magnetite nanopart
application
Scheme
Scheme as particulate
5. Covalent
5. Covalent vaccines
immobilization
immobilization of[50].
of enzymes to CNBr-agarose.
enzymes to CNBr‐agarose.

Different immobilization binding chemistry can be used when

carbonyldiimidazole (CDI) is employed to activate the agarose. CDI is a highly
carboxylating agent that contains two acylimidazole leaving groups, which form
carbonyl groups on the hydroxyl support. This conjugate reagent has been succ

Scheme
Scheme Covalent
6. 6. immobilization
Covalent of enzymes
immobilization to CDI-agarose.
of enzymes to CDI‐agarose.
The same binding chemistry described for immobilizing enzymes on different func-
Thecarriers
tionalized sameisbinding chemistry
used for enzyme described
immobilization for immobilizing
on membranes. In this case, enzymes
chemical on d
functionalized
bonds carriers
can be present eitherisonused for enzyme
the surface or withinimmobilization
the pores of theon membranes.
membrane. In In th
most membranes,
chemical bondsthere
can are
be few naturally
present occurring
either on thefunctional
surface groups
or withinthat the
can be directly
pores of the me
used for covalent bonding. For this reason, membrane pretreatment with functionalizing
In most membranes, there are few naturally occurring functional groups that
reagents is commonly applied for covalent immobilization using methods such as wet
directly used for covalent bonding. For this reason, membrane pretreatme
chemistry, UV activation, gamma irradiation, and plasma treatment. A notable example is
functionalizing
β-galactosidase reagents
covalently is commonly
attached applied for
to polyethersulfone covalentfunctionalized
membranes, immobilization with using m
suchforas
GA, thewet chemistry,
continuous UVofactivation,
hydrolysis lactose in the gamma irradiation,
dairy industry. This system and plasma
enables the treatm
production of lactose-free
notable example milk with an extended
is β‐galactosidase enzyme lifespan.
covalently attached However, the rigidity of mem
to polyethersulfone
covalent bonds may induce conformational changes in the enzyme, potentially reducing its
functionalized with GA, for the continuous hydrolysis of lactose in the dairy in
catalytic efficiency. Another example is laccase immobilized on TiO2 -coated membranes
This system
activated with enables
APTES and thethen
production
GA (activityof lactose‐free milkused
recovery of 79%), withinan extended enzyme l
pharmaceutical
However,treatment.
wastewater the rigidity of covalent
The enzyme bonds
is chemically may
linked to theinduce
modifiedconformational
membrane surface,changes
enzyme,forpotentially
allowing reducing
long-term degradation its catalytic
of antibiotics such asefficiency.
erythromycinAnother example is
and tetracyclines.
This immobilization strategy ensures minimal enzyme
immobilized on TiO2‐coated membranes activated with APTES and thenloss, making it highly effective in GA
continuous processes [26].
recovery of 79%), used in pharmaceutical wastewater treatment. The enz
chemically
3. linked
Site-Specific to the modified membrane surface, allowing for long‐term degr
Immobilization
of antibiotics such as an
For some applications, erythromycin and tetracyclines.
oriented immobilization This desirable.
protocol is highly immobilization
In
ensures minimal
biocatalysis, enzyme loss,
optimal accessibility making
of the substrateitto
highly effective
the active in continuous
site of immobilized processes
enzymes
is highly sought. Being able to immobilize the biocatalyst with its active site oriented
toward the solution through an oriented immobilization protocol allows higher catalytic
3. Site‐Specific Immobilization
efficiency and a homogeneous activity to be obtained compared to an immobilized biocata-
lyst byFor some applications,
a conventional an oriented
method, leading immobilization
to a randomly protocolIniscontrast,
desir
oriented immobilization. highly
biocatalysis, optimal accessibility of the substrate to the active site of imm
enzymes is highly sought. Being able to immobilize the biocatalyst with its ac
oriented toward the solution through an oriented immobilization protocol allow
catalytic efficiency and a homogeneous activity to be obtained compared
Molecules 2025, 30, 939 12 of 33

redox enzymes generally require their active site to be in close contact with the support
surface to optimally receive or transfer electrons from or to the support [65]. The ability to
form an oriented binding relies on unique chemical functionalities present on the surface
of the enzyme that has to be immobilized. These unique chemical functionalities can be
naturally present or synthetically introduced by engineering approaches into the amino
Molecules 2025, 30, x FOR PEER REVIEW 13 of 35
acidic sequence of the enzyme. Few examples of naturally unique reactive groups occurring
are reported in the literature. For example, glycosylated enzymes are characterized by the
presence on their surface of covalently bound oligosaccharide units. These carbohydrate
bound oligosaccharide
residues, if properly exposed units.on These carbohydrate
the surface residues,
of the enzyme and if if
properly exposed
not essential on cat-
for the the
alytic activity, can be used for a reversible covalent interaction with boronic acid derivatizeda
surface of the enzyme and if not essential for the catalytic activity, can be used for
reversible
carriers covalent
[66]. Different interaction
classes of with boronic
enzymes acidbeen
have derivatized
immobilized carriersvia[66]. Different
boronic classes
acid-cis-diol
of enzymes have been immobilized via boronic acid‐cis‐diol
interaction: horseradish peroxidase [67], lipase from Candida antarctica B [68], laccase from interaction: horseradish
peroxidase
Pleurotus [67], lipase
ostreatus from Candida
[69]. Other examples antarctica
rely on B [68],
the laccaseoffrom
presence uniquePleurotus
amino ostreatus
acid residues[69].
Other examples rely on the presence of unique amino acid
in the enzyme sequence. Pepsin contains one phosphoserine residue in its whole sequence residues in the enzyme
sequence.
(304 aminoPepsinacids). contains
This uniqueone residue
phosphoserine residueexploited
was selectively in its whole sequence
for its (304 amino
immobilization on
acids). This unique residue was selectively exploited for
alumina [70]. Bromelain contains just one His residue (His158) in its whole amino its immobilization on alumina
acidic
[70]. Bromelain
sequence (23.8 kDa).contains
Thisjust
uniqueone residue
His residuewas (His158)
successfully in its whole amino
exploited for its acidic sequence
immobilization
(23.8 kDa).
2+ This unique residue was successfully exploited
on Cu derivatized iminodiacetic acid Sepharose 6B carrier [71]. However, since for its immobilization on Cu the
2+

derivatized
presence iminodiacetic
of naturally acid Sepharose
occurring 6B carrier
unique residues [71].rare,
is quite However,
nowadays,since the
the sequence
presence of of
naturally occurring unique residues is quite rare, nowadays, the
the enzymes can be easily engineered by the addition of tags at the N- or C-terminus or by sequence of the enzymes
can introduction
the be easily engineered by the addition
of unique chemical of tags
functionalities intoatthe thesequence
N‐ or C‐terminus
of the protein ortoby the
allow
introduction of unique chemical functionalities into the
a site-specific immobilization protocol. The possible engineering techniques can includea
sequence of the protein to allow
site‐specific
the fusion ofimmobilization
the N- or C-terminus protocol. The
of the possible
protein engineering
sequence techniques
to genetically can include
encoded functionalthe
fusion of the N‐ or C‐terminus of the protein sequence to genetically
tags [72], the use of post-translational enzyme-catalyzed protein modifications [73], the encoded functional
tags [72], the use
incorporation of post‐translational
of naturally occurring amino enzyme‐catalyzed
acids by site-specific protein modifications
mutagenesis into[73], the
a small
incorporation of naturally occurring amino acids by site‐specific
area of the protein surface [65], the incorporation of unique unnatural amino acids into mutagenesis into a small
area of sequences
protein the proteinbysurfacegenetic[65], the incorporation
methods such as amber ofcodon
unique unnatural mutagenesis
suppression amino acids [74]. into
protein sequences by genetic methods such as amber codon suppression
In this section, we will discuss in more detail the use of engineering techniques reported mutagenesis [74].
In the
in thisliterature
section, we in will
orderdiscuss
to achievein more detail thesite-specific
an oriented use of engineering techniques
immobilization reported
protocol for
in the literature in order to achieve an oriented site‐specific
enzymes applied in biocatalysis. The site-specific immobilization approach can be dividedimmobilization protocol for
enzymes
into applied in
non-covalent andbiocatalysis. The site‐specific
covalent strategies (Figure immobilization
2). approach can be divided
into non‐covalent and covalent strategies (Figure 2).

Figure
Figure 2. Site-specific immobilization techniques classification.
2. Site‐specific classification.

3.1. Site‐Specific Non‐Covalent Immobilization

Enzymes can be non‐covalently bound in an oriented way onto a carrier via affinity
interaction. The enzyme sequence must be engineered using recombinant techniques in
order to introduce an affinity tag at the N‐ or C‐terminus of the protein sequence that is
able to recognize and bind specifically to its counterpart present on the immobilization
carrier. Affinity immobilization techniques exploit the selectivity of specific interactions
such as between polyhistidine and metal ions, (strept)avidin and biotin, antibodies and
antigens, lectins and glycosylated macromolecules, nucleic acids and nucleic acid‐binding
Molecules 2025, 30, 939 13 of 33

3.1. Site-Specific Non-Covalent Immobilization

Enzymes can be non-covalently bound in an oriented way onto a carrier via affinity
interaction. The enzyme sequence must be engineered using recombinant techniques in
order to introduce an affinity tag at the N- or C-terminus of the protein sequence that is able
to recognize and bind specifically to its counterpart present on the immobilization carrier.
Affinity immobilization techniques exploit the selectivity of specific interactions such as
between polyhistidine and metal ions, (strept)avidin and biotin, antibodies and antigens,
Molecules 2025, 30, x FOR PEER REVIEW 14 of 35
lectins and glycosylated macromolecules, nucleic acids and nucleic acid-binding proteins,
hormones, and their receptors and many more [72]. However, these selective non-covalent
immobilization strategies
purification area. havebinding
Therefore, mostly been borrowed
is usually from themaking
reversible, protein them
purification area.
sometimes
Therefore, binding is usually reversible, making them sometimes inappropriate as an
inappropriate as an effective immobilization technique.
effective immobilization technique.
3.1.1. Site‐Specific Non‐Covalent Immobilization via Metal Affinity
3.1.1. Site-Specific Non-Covalent Immobilization via Metal Affinity
A versatile and widely used affinity tag is the polyhistidine tag (His‐tag). The
A versatile and widely used affinity tag is the polyhistidine tag (His-tag). The polyhis-
polyhistidine tag (6 to 10 histidine residues, typically) can be easily fused to the N‐ or C‐
tidine tag (6 to 10 histidine residues, typically) can be easily fused to the N- or C-terminus
terminus of the enzyme of interest thanks to the availability of several commercial
of the enzyme of interest thanks to the availability of several commercial expression vectors
expression vectors that include this tag, and the carrier has to be derivatized with
that include this tag, and the carrier has to be derivatized with chelating moieties (such as
chelating moieties (such as nitrilotriacetic acid or iminodiacetic acid) bearing divalent
nitrilotriacetic acid or iminodiacetic acid) bearing divalent metal ions (Cu2+ , Ni2+ , Zn2+ ,
metal ions (Cu2+, Ni2+, Zn2+, Co2+) or trivalent metal ions (Fe3+). The choice of metal ions to
Co2+ ) or trivalent metal ions (Fe3+ ). The choice of metal ions to be used for carrier derivati-
be used for carrier derivatization is highly dependent on the application since the different
zation is highly dependent on the application since the different metal ions have different
metal ions have different affinity and specificity in binding His‐tagged proteins.
affinity and specificity in binding His-tagged proteins.
This technique is widely used in the biocatalysis field to achieve a one‐pot
This technique is widely used in the biocatalysis field to achieve a one-pot purification
purification and immobilization protocol of the desired recombinant enzyme. Many
and immobilization protocol of the desired recombinant enzyme. Many enzymes have
enzymes have been immobilized via His‐tag both for biocatalytic application and
been immobilized via His-tag both for biocatalytic application and biosensor development
biosensor development on different carrier materials: cutinase [75], alcohol oxidase [76],
on different carrier materials: cutinase [75], alcohol oxidase [76], ω-transaminases [77],
‐transaminases [77], ene‐reductases [78], ‐galactosidase [79], ‐mannosidase [80]. The
ene-reductases [78], β-galactosidase [79], β-mannosidase [80]. The commercially available
commercially available EziGTM carriers by EnginZyme AB Sweden (Stockholm, Sweden)
EziGTM carriers by EnginZyme AB Sweden (Stockholm, Sweden) have been used for the
have been used for the immobilization of a broad range of enzymes for both batch and
immobilization of a broad range of enzymes for both batch and flow biocatalytic applica-
flow biocatalytic applications [76,81]. These carriers are based on glass beads (plastic‐free)
tions [76,81]. These carriers are based on glass beads (plastic-free) coated with an organic
coated with an organic polymer and chelated Fe3+ ions, which enable the selective binding
polymer and chelated Fe3+ ions, which enable the selective binding of His-tagged proteins
of His‐tagged proteins directly from crude cell lysate allowing to achieve a one‐pot
directly from crude cell lysate allowing to achieve a one-pot purification and immobilization
purification and immobilization protocol (Scheme 7). EziGTM is available in three versions
protocol (Scheme 7). EziGTM is available in three versions with different surface hydropho-
with different
bicity: surface hydrophobicity:
Opal (hydrophilic), Opal (hydrophilic),
Coral (hydrophobic), Coral (hydrophobic),
and Amber (semi-hydrophilic) [76]. and
Amber (semi‐hydrophilic) [76].

Scheme 7. Immobilization of His6 -tagged enzyme onto commercial EziGTM support. The enzyme is
Scheme 7. Immobilization of His6‐tagged enzyme onto commercial EziGTM support. The enzyme is
immobilized onto the solid carrier through chelation of the metal ion by the imidazole moieties of the
immobilized onto the solid carrier through chelation of the metal ion by the imidazole moieties of
His residues.
the His residues.

As already said, the fusion of histidine tags can only occur to the N‐ or C‐terminus of
recombinant proteins, limiting the possibilities of enzyme orientation. In a recent example,
the surface of two dehydrogenases (i.e., ADH from Bacillus stearothermophilus and ADH
from Thermus thermophilus) was engineered by adding His‐clusters into regions not
Molecules 2025, 30, 939 14 of 33

As already said, the fusion of histidine tags can only occur to the N- or C-terminus of
recombinant proteins, limiting the possibilities of enzyme orientation. In a recent example,
the surface of two dehydrogenases (i.e., ADH from Bacillus stearothermophilus and ADH
from Thermus thermophilus) was engineered by adding His-clusters into regions not involved
in the catalysis in order to drive the immobilization via coordination bond [82]. By varying
the position and the histidine density of the clusters, a small library of enzyme variants
Molecules 2025, 30, x FOR PEER REVIEW
was created and immobilized on different carriers functionalized with different densities 15 ofof
35
2+ 2+ 2+ 3+
various metal chelates (Co , Cu , Ni , and Fe ). The authors demonstrated that His-
clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering
even
somemore activity, and
His‐clustered gainingcertain
variants, stabilityorientations
against some denaturing
lead to more agents. For some His-
active heterogeneous
clustered variants,
biocatalysts, whereascertain
otherorientations
orientations lead
leadto tomore
more active heterogeneous
stable immobilizedbiocatalysts,
enzymes in
whereas othertoorientations
comparison lead tocounterparts
their His‐tagged more stable immobilized
immobilizedenzymes on the samein comparison
carriers. to their
His-tagged
However,counterparts immobilized
the coordination on the same
interaction carriers. and can be disrupted by the
is reversible
However,
addition the coordination
of competitive interaction
ligands is reversibleorand
like imidazole bycan be disrupted
metal chelatorsby(e.g.,
the
addition of competitive ligands
ethylenediaminetetraacetic acid).like
Theimidazole
reversibility orof bythemetal chelators
binding can be(e.g., ethylenedi-
an advantage for
aminetetraacetic
certain applicationsacid).where
The reversibility
reusability of the binding carrier is canrequired
be an advantage for certain
(e.g., purification of
applications
recombinantwhere reusability
proteins from celloflysates
the carrier is required
or biochips (e.g., purification
production) but can beof recombinant
a disadvantage
proteins from
if stability andcell lysates
shelf or biochips
life are an issueproduction)
for the desired but can
finalbeimmobilized
a disadvantage if stability
derivatives. To
and shelf the
enhance life stability
are an issue
of theforbinding,
the desired finalinteraction
His‐tag immobilized canderivatives.
be combined Towith
enhance the
covalent
stability
bindingof the binding,
through His-tag
classical interaction can
immobilization be combined
by designing with covalent binding
a heterofunctional carrier through
(Scheme
classical immobilization
8). In this case, a two‐step by immobilization
designing a heterofunctional
process will occur: carrierfirst,
(Scheme 8). In this
the protein case,
will comea
two-step immobilization
in close contact with the process
carrier inwill
anoccur:
orientedfirst,
waythebyprotein will
affinity come in
His‐tag close contact
interaction with
followed
the
by carrier
covalent inbond
an oriented
formationwaywithby affinity
classicalHis-tag
chemical interaction followed
groups present onbythecovalent
surface bond
of the
formation with
carrier (e.g., classical
epoxy chemical
groups). groups
[61,83]. More present on the
recently, a newsurface of thesite‐specific
covalent carrier (e.g.,method
epoxy
groups).
was also[61,83]. Morefor
developed recently, a newproteins
His‐tagged covalentonto site-specific method derivatized
a vinyl sulfone was also developed for
surface (see
His-tagged
later) [84]. proteins onto a vinyl sulfone derivatized surface (see later) [84].

Scheme
Scheme 8. ImmobilizationofofHis
8. Immobilization His 6 -tagged
6‐tagged enzyme
enzyme by by mixed
mixed binding
binding chemistry.
chemistry. Preparation
Preparation of
of metal
metal chelate
chelate epoxy epoxy carrier:
carrier: commercially
commercially available
available methacrylic
methacrylic epoxycarrier
epoxy carrier isis derivatized
derivatized with
with
iminodiacetic acid to introduce a few metal chelate sites. The IDA carrier is then incubated
iminodiacetic acid to introduce a few metal chelate sites. The IDA carrier is then incubated with with
cobalt
cobaltsalts
saltstotoobtain
obtainmetal
metalchelate
chelateepoxy
epoxycarriers.
carriers.The
Theenzyme
enzymecomes
comesfirst
firstin
inclose
closecontact
contactwith
withthe
the
carrier through affinity interaction. Afterward, immobilization conditions are changed in order to
carrier through affinity interaction. Afterward, immobilization conditions are changed in order to
allow the covalent bond formation between the remaining epoxy groups of the carrier and “classical”
allow the covalent bond formation between the remaining epoxy groups of the carrier and
amino-bearing groups present on the enzyme surface (e.g., lysins).
“classical” amino‐bearing groups present on the enzyme surface (e.g., lysins).
3.1.2. Site-Specific Non-Covalent Immobilization via Biotin-(Strept)Avidin Interaction
3.1.2. Site‐Specific Non‐Covalent Immobilization via Biotin‐(Strept)Avidin Interaction
The biotin-(strept)avidin interaction is one of the most specific and stable non-covalent
The biotin‐(strept)avidin
interactions with a dissociationinteraction is one
constant about 103ofto the
106 most
times specific and an
higher than stable non‐
epitope–
covalent interaction.
antibody interactionsBiotinylated
with a dissociation
enzymes constant about 10 toonto
can be immobilized 3 10 (strept)
6 times higher than an
avidin-coated
surfaces. To perform this immobilization protocol, the enzyme to be immobilized(strept)
epitope–antibody interaction. Biotinylated enzymes can be immobilized onto has to
avidin‐coated
be engineered in surfaces.
order toTo performonthis
introduce its immobilization
surface the biotin protocol,
moiety. the enzyme
At the same to be
time
immobilized has to be engineered in order to introduce on its surface the
the carrier has to be engineered and derivatized with (strept)avidin (53 kDa). Biotiny- biotin moiety.
At the
lated same time
enzymes can the carrier has
be prepared to be engineered
through and derivatized
chemical methods with (strept)avidin
that generally conjugate biotin(53
kDa). Biotinylated enzymes can be prepared through chemical methods that generally
conjugate biotin molecules to Lys residues of the protein. Pullulanese, an important
biocatalyst in the food industry, was immobilized onto magnetic nanoparticles based on
the specific recognition between biotin and streptavidin. Pullulanase was chemically
 Molecules 2025, 30, 939 15 of 33

molecules to Lys residues of the protein. Pullulanese, an important biocatalyst in the food
industry, was immobilized onto magnetic nanoparticles based on the specific recognition
between biotin and streptavidin. Pullulanase was chemically biotinylated using biotin-N-
hydroxysuccinimide, and the magnetic nanoparticles were prepared and functionalized
Molecules 2025, 30, x FOR PEER REVIEWwith streptavidin using cyanuric chloride. The immobilized pullulanase retained 16 ofhigh
35
levels of activity (85%) and exhibited significantly improved pH and thermal stability
as compared to the soluble enzyme. Moreover, the excellent recyclability of the immo-
bilized
its initialbiocatalyst was eight
activity after also reported,
consecutiveretaining
reactionmore than[85].
cycles 74% Inof its initialexample,
another activity after
β‐
eight consecutive reaction cycles [85]. In another example, β-agarase
agarase was chemically biotinylated through two different activation procedures: (i) the was chemically bi-
otinylated through two different activation procedures: (i) the amino
amino groups of ‐agarase were reacted with N‐succinimidyl 6‐biotinamidohexanoate, (ii) groups of β-agarase
were
the carboxyl with N-succinimidyl
reactedgroups of ‐agarase were6-biotinamidohexanoate, (ii) the carboxyl groups of β-
first reacted with 1‐(3‐dimethylaminopropyl)‐3‐
agarase were first reacted with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
ethylcarbodiimide hydrochloride and N‐hydroxysuccinimide to obtain amine‐reactive hydrochlo-
ride and N-hydroxysuccinimide
terminus and, then coupled with to biotin
obtain amine-reactive
C‐5‐amine (Scheme terminus and, thenshowed
9). Results coupledthat,
with
biotin C-5-amine (Scheme 9). Results showed that, compared with
compared with the soluble enzyme, the stability and substrate affinity of immobilized β‐ the soluble enzyme,
the stability
agarase and substrate
preparations affinity
through of immobilized
amino or carboxylβ-agarase
activationpreparations
were boththrough amino
significantly
or carboxyl activation were both significantly improved.
improved. However, the amino‐activated immobilized β‐agarase showed higher However, the amino-activated
immobilized β-agarase
thermostability showed
and catalytic higher thermostability
efficiency and catalytic efficiency
than the carboxyl‐activated immobilizedthan β‐
the
carboxyl-activated
agarase [86]. immobilized β-agarase [86].

Scheme
Scheme 9. Immobilization
Immobilizationof of ‐agarase
β-agarase through
through biotin‐streptavidin
biotin-streptavidin interaction.
interaction. Magnetic
Magnetic nanopar-
ticles Fe3 O4 (MNP)
nanoparticles were prepared
Fe3O4 (MNP) using the using
were prepared solvothermal reduction method
the solvothermal andmethod
reduction subsequently
and
derivatized with
subsequently cyanuric acid
derivatized with(NCCl) 3 andacid
cyanuric streptavidin
(NCCl)3 (SA).
and β-Agarase was(SA).
streptavidin ‐Agarase
chemically derivatized
was
with biotinderivatized
chemically through itswith
amino or carboxyl
biotin throughgroups.
its amino or carboxyl groups.

One limitation in applying a chemical biotinylation procedure is the difficulty in

One limitation in applying a chemical biotinylation procedure is the difficulty in
controlling both the extent and the site of biotinylation since most proteins have more
controlling both the extent and the site of biotinylation since most proteins have more
than one residue on their surface that can be biotinylated. Thus, the overall orientation of
than one residue on their surface that can be biotinylated. Thus, the overall orientation of
the immobilized enzyme cannot be precisely controlled, but it depends on which biotin(s)
the immobilized enzyme cannot be precisely controlled, but it depends on which biotin(s)
interacts with the strept(avidin) probe. Nowadays, it is possible to apply a controlled
interacts with the strept(avidin) probe. Nowadays, it is possible to apply a controlled
biotinylation procedure by fusing the N- or C-terminus of the enzyme that has to be
biotinylation procedure by fusing the N‐ or C‐terminus of the enzyme that has to be
immobilized with a 75 amino acid biotin carboxyl carrier protein (BCCP) module from
immobilized with a 75 amino acid biotin carboxyl carrier protein (BCCP) module from E.
E. coli acetyl CoA carboxylase [87]. The BCCP tag is recognized and can be biotinylated
coli acetyl CoA carboxylase [87]. The BCCP tag is recognized and can be biotinylated in
in vivo or in vitro by E. coli biotin ligase/synthetase (BirA). The ligation reaction catalyzed
vivo or in vitro by E. coli biotin ligase/synthetase (BirA). The ligation reaction catalyzed
by BirA can also be applied for the biotinylation of other smaller tags such as the Avitag, a
by BirA can also be applied for the biotinylation of other smaller tags such as the Avitag,
tag of 15 amino acids that is rapidly biotinylated by BirA [88]. β-Galactosidase from E. coli
a tag of 15 amino acids that is rapidly biotinylated by BirA [88]. ‐Galactosidase from E.
was fused at its N-terminus with a polypeptide tag that was site-specifically biotinylated
coli was fused at its N‐terminus with a polypeptide tag that was site‐specifically
by biotin ligase during the post-translational modification process in E. coli. Subsequently,
biotinylated by biotin ligase during the post‐translational modification process in E. coli.
the biotinylated enzyme was incubated in the presence of poly(ether sulfone) membranes
Subsequently, the biotinylated enzyme was incubated in the presence of poly(ether
derivatized with avidin. The activity of this site-specific immobilized enzyme was two-fold
sulfone) membranes derivatized with avidin. The activity of this site‐specific immobilized
higher compared to that of the commercially available non-specific biotinylated enzyme
enzyme was two‐fold higher compared to that of the commercially available non‐specific
biotinylated enzyme that was immobilized in a random way on the same carrier [89]. The
C‐terminus of ‐glucosidase from B. licheniformis was fused with a biotin acceptor peptide
and co‐expressed with BirA biotin ligase. The immobilized enzyme showed improved
thermal stability compared to the soluble enzyme and good recyclability (after nine cycles
Molecules 2025, 30, 939 16 of 33

that was immobilized in a random way on the same carrier [89]. The C-terminus of β-
glucosidase from B. licheniformis was fused with a biotin acceptor peptide and co-expressed
with BirA biotin ligase. The immobilized enzyme showed improved thermal stability
compared to the soluble enzyme and good recyclability (after nine cycles of reaction, the
biocatalyst could retain 89% of its initial activity) [90].

3.2. Site-Specific Covalent Immobilization

The site-specific covalent immobilization procedures rely on the introduction of a
unique bio-orthogonal chemical group or a sequence tag in the enzyme at a site-specific
location that is able to form a covalent bond with a mutually reactive group present on
a carrier. The site of enzyme engineering has to be strategically chosen in order to not
impact the conformation of the enzyme and, thus, its catalytic activity. Three different
strategies can be applied to achieve site-specific covalent immobilization as depicted in
Figure 2: (i) tag-mediated immobilization, (ii) immobilization via naturally occurring amino
acids positioned in specific regions of the protein, and (iii) immobilization via unnatural
amino acids.

3.2.1. Tag-Mediated Bio-Orthogonal Covalent Immobilization

Covalently binding tags allow a rapid, highly specific, and irreversible binding of
recombinantly tagged proteins to specific ligands that can be covalently or non-covalently
attached to the solid carriers. As depicted in Figure 2, covalent immobilization tag sys-
tems can be based on (i) classical small peptide tags (His-tag, Cys-tag); (ii) self-labeling
protein tags constituted by enzymes that are able to catalyze their covalent attachment to
specific synthetic ligands (HaloTag (Promega), SNAP-tag (New England Biolabs), CLIP-
tag (New England Biolabs)); (iii) bacterial domains containing intramolecular isopeptide
bonds split into two parts, a Tag peptide, and a Catcher protein; and (iv) small peptides
that are recognized in vitro by enzymes able to mediated a bond formation (e.g., sortase,
transglutaminase). The engineering of enzymes by fusing a specific tag sequence can
occur just to the N- or C-terminus of recombinant proteins, limiting the possibilities of
enzyme orientation. However, generally, these tags are small molecules that do not affect
the expression and solubility of the engineered enzymes, and sometimes, these tags can
also enhance the solubility of recombinant proteins. Moreover, the fusion of tags does not
require the availability of the three-dimensional structure of the protein.

Classical Small Peptide Tags

As already anticipated, a new covalent site-specific method was developed for
the immobilization of proteins through His-tag [84]. Two model His-tagged proteins
(HaloTag-6xHis and anti-HER2 Fab-6xHis) were conjugated onto surfaces derivatized with
vinyl sulfone groups through a covalent bond (Scheme 10). Other than covalent bind-
ing, the newly developed protocol allowed us to obtain high protein loading and high
binding specificity.
Enzymes with a tag consisting of cysteine repeats (Cys-tag) genetically fused at one
terminus can be covalently attached to a solid support by disulfide bond formation. Purine
nucleoside phosphorylase from Halomonas elongata (HePNP) with a Cys6 -tag was im-
mobilized on agarose beads activated with thiol (SH) groups (Scheme 11) and used for
the synthesis of nucleosides analogs in the flow system [91]. The resulting immobilized
HePNP on SH-agarose was shown to be as active as the enzyme covalently immobilized on
epoxy/Co2+ -activated agarose and more active than HePNP immobilized on other carriers
(silica or methacrylate) with different binding chemistries (epoxy/Co2+ , aldehyde). This
immobilization strategy allowed an oriented and selective immobilization protocol to be
obtained compared to the other tested immobilization strategies. Moreover, the covalent
 require the availability of the three‐dimensional structure of the protein.

Classical Small Peptide Tags

Molecules 2025, 30, 939 As already anticipated, a new covalent site‐specific method was developed 17 forofthe
33
immobilization of proteins through His‐tag [84]. Two model His‐tagged proteins
(HaloTag‐6xHis and anti‐HER2 Fab‐6xHis) were conjugated onto surfaces derivatized
but reversible
with binding
vinyl sulfone chemistry
groups allowed
through the reusability
a covalent of expensive
bond (Scheme agarose
10). Other beads
than in a
covalent
second cycle of enzyme immobilization after inactive enzyme removal with DTT treatment
binding, the newly developed protocol allowed us to obtain high protein loading and high
(50 mM). specificity.
binding
Molecules 2025, 30, x FOR PEER REVIEW 18 of 35

Scheme 10. Covalent site‐specific immobilization of His‐tagged proteins onto vinyl sulfone‐bearing
surface. The imidazole group of the last His residue of His6‐tag is deprotonated at neutral pH and
can undergo Michael‐type addition by vinyl sulfone groups present onto the carrier.

Enzymes with a tag consisting of cysteine repeats (Cys‐tag) genetically fused at one
terminus can be covalently attached to a solid support by disulfide bond formation.
Purine nucleoside phosphorylase from Halomonas elongata (HePNP) with a Cys6‐tag was
immobilized on agarose beads activated with thiol (SH) groups (Scheme 11) and used for
the synthesis of nucleosides analogs in the flow system [91]. The resulting immobilized
HePNP on SH‐agarose was shown to be as active as the enzyme covalently immobilized
on epoxy/Co2+‐activated agarose and more active than HePNP immobilized on other
carriers (silica or methacrylate) with different binding chemistries (epoxy/Co2+, aldehyde).
This immobilization strategy allowed an oriented and selective immobilization protocol
to be obtained
Scheme compared
10. Covalent to the
site-specific other tested
immobilization of immobilization strategies.
His-tagged proteins onto vinylMoreover, the
sulfone-bearing
covalent but reversible binding chemistry allowed the reusability of expensive agarose
surface. The imidazole group of the last His residue of His6 -tag is deprotonated at neutral pH and
beads in a second
can undergo cycle addition
Michael-type of enzyme immobilization
by vinyl sulfone groupsafter inactive
present enzyme
onto the carrier.removal with
DTT treatment (50 mM).

Scheme 11.
Scheme Covalent site‐specific
11. Covalent site-specific immobilization
immobilization of
of Cys‐tagged
Cys-tagged proteins
proteins onto
onto thiol
thiol agarose. The
The
commercially available
commercially available epoxy
epoxy agarose
agarose was
was derivatized
derivatized with
with the
the thiol
thiol group
group in
in two
two steps:
steps: overnight
overnight
incubation with
incubation with 50
50 mM
mM Na Na22SSin
in 100
100 mM
mM NaHCO
NaHCO33pH pH1010and
and subsequently
subsequentlyincubation
incubationofof33 hh with
with
10 mM 2,2 ′ -dinitro-5,5′ -thiobenzoic acid (DTNB). The Cys-tagged enzyme was covalently bound to
10 mM 2,2′‐dinitro‐5,5′‐thiobenzoic acid (DTNB). The Cys‐tagged enzyme was covalently bound to
the carrier by thiol exchange.
the carrier by thiol exchange.
Self-Labeling Protein Tags
Self‐Labeling Protein Tags
Self-labeling protein tags are small proteins, typically less than 40 kDa, able to catalyze
Self‐labeling protein tags
the self-covalent attachment to are small proteins,
a small-molecule typically
probe that isless than 40 kDa,
functionalized able
with to
a bio-
catalyze the self‐covalent attachment to a small‐molecule probe that is functionalized
orthogonal linker [92]. Self-labeling protein tags include the HaloTag (33 kDa), SNAP-tag with
a(22
bio‐orthogonal linker an
kDa), and CLIP-tag, [92]. Self‐labeling
engineered protein
variant of thetags include(Scheme
SNAP-tag the HaloTag
12). (33 kDa),
SNAP‐tag (22 kDa), and CLIP‐tag, an engineered variant of the SNAP‐tag (Scheme 12).
Molecules2025,
Molecules 2025,30,
30,939
x FOR PEER REVIEW 19 of 35
18 of 33

Scheme12.
Scheme 12.Site-specific
Site‐specificcovalent
covalentenzyme
enzymeimmobilization
immobilization by
byfusion
fusionto
toself-labeling
self‐labelingtags:
tags:HaloTag
HaloTag
(33kDa)
(33 kDa)forms
formsaacovalent
covalentbond
bondthrough
throughnucleophilic
nucleophilicdisplacement
displacement of
ofhalides
halidesfrom
fromalkyl
alkylhalide
halide
derivatizedcarriers,
derivatized carriers,SNAP-tag
SNAP‐tag(20
(20kDa)
kDa)reacts
reactswith
withOO6 -benzylguanine
6‐benzylguaninederivatized
derivatizedcarriers
carrierswhile
while
CLIP-tag 2
CLIP‐tag(also
(also20
20kDa)
kDa)reacts
reactswith
withOO-benzylcytosine
2 ‐benzylcytosinederivatized
derivatizedcarriers.
carriers.

Among the tags based on enzymes that are able to catalyze their covalent attachment
to specific synthetic ligands, the HaloTagTM is the most used for enzyme immobilization
Molecules 2025, 30, 939 19 of 33

Among the tags based on enzymes that are able to catalyze their covalent attachment to
specific synthetic ligands, the HaloTagTM is the most used for enzyme immobilization [72].
This system is based on a modified haloalkane dehalogenase from Rhodococcus rhodocrous
that specifically recognizes terminal chloroalkane ligands, with which it establishes a sta-
ble covalent ester bond via an aspartate residue present in the active site (Scheme 12).
The thiamine diphosphate-dependent benzaldehyde lyase from Pseudomonas fluorescence
(PfBAL) fused with HaloTag was rapidly purified and immobilized from crude extracts
in high purity. The immobilized biocatalyst was stable at 4 ◦ C for months and was suc-
cessfully reused in several repetitive batches for the carboligation of aggressive aldehydes
(benzaldehyde and acetaldehyde) toward (R)-hydroxy-1-phenylpropan-1-one [93]. Using
the same strategy, benzoylformate decarboxylase from Pseudomonas putida (PpBFD) was
combined with alcohol dehydrogenase from Lactobacillus brevis (LbADH) in a continuous
enzymatic cascade. Both enzymes were selectively purified and immobilized on Halolink
ResinTM from crude cell extracts directly in plug-flow reactors. The system resulted in the
formation of the target product (1S,2S)-1-phenylpropane-1,2-diol with high conversion
rates and stereoselectivity (up to 99% and 96%, respectively), and high space-time yields
(up to 1850 g L−1 d−1 ) [94]. Recently, three Fe(II)/α-ketoglutarate-dependent dioxygenases
(CaKDO, CpKDO, FjKDO) were easily purified from cell lysates and covalently immo-
bilized by the HaloTag system [95]. The recombinant production and immobilization of
KDO are quite challenging due to their low solubility during expression and instability
once purified. The HaloTag-based immobilization allowed a rapid concentration of the
enzymes from cell-free extracts on the carrier surface with high residual activity and im-
proved stability. The increase in the stability enabled the desired biotransformation to be
performed using high substrate loading without the generation of any side products and
the recycling of the biocatalysts. Moreover, the immobilized biocatalysts were also used in
preparative lab-scale biotransformations, achieving product titers of 16 g L−1 (3S)-hydroxy-
L-lysine (CaKDO) and (4R)-hydroxy- L-lysine (FjKDO), respectively, starting from 100 mM
L-lysine. Additionally, a Halo-tagged immobilized lysine decarboxylase from Selenomonas
ruminantium was utilized to convert the (3S)-hydroxy-L-lysine produced by CaKDO into
(2S)-hydroxy-cadaverine in a 15 mL consecutive batch reaction and without intermediate
product purification.

Catcher/Tag Systems
Another emerging immobilization approach in biocatalysis is the SpyTag/SpyCatcher
system. The SpyTag/SpyCatcher pair is derived from the splitting and engineering of
the CnaB2 domain of the fibronectin-binding protein (FbaB) from Streptococcus pyogenes.
The SpyTag is constituted by the C-terminal beta strand of CnaB2 (13 aa) containing a
reactive aspartic acid (Asp117), while the SpyCatcher (116 aa) is constituted by the rest
of the beta strands containing a reactive lysine (Lys31). When the tag, generally fused
to the recombinant enzyme or sometimes present onto the carrier, comes in close contact
with its corresponding catcher, present on the carrier surface or fused to the recombinant
enzyme, the interrupted intramolecular isopeptide bond is spontaneously reconstituted by
forming a stable covalent bond (Scheme 13) [96]. The original SpyTag/SpyCatcher pair was
further improved in order to achieve faster reaction rates (SpyTag002/3:SpyCatcher002/3)
when compared to the original pair [97]. Furthermore, other Tag/Catcher pairs were
developed by finding orthogonal proteins from different bacterial species, such as the
SnoopTag/SnoopCatcher [98] and the SdyTag/SdyCatcher [99]. Additionally, an engi-
neered non-reactive SpyCatcher variant (SpyDock) was developed in order to allow an
on-demand reversible affinity binding [100]. For now, this system, called Spy&Go, has
Molecules 2025, 30, 939 20 of 33

been
Molecules 2025, 30, x FOR PEER REVIEW employed for the purification of SpyTagged proteins, but it could also be exploited
21 of 35
for enzyme immobilization.

Scheme
Scheme13. 13. Site-specific
Site‐specificcovalent
covalentenzyme
enzymeimmobilization
immobilizationby
bySpyTag/SpyCatcher.
SpyTag/SpyCatcher. TheThe formation
formationof
of
the
theisopeptide
isopeptidebond
bondbetween
betweenaa lysine
lysine in
in SpyCatcher
SpyCatcher (Lys31)
(Lys31) and
and an
an aspartic
aspartic acid in SpyTag
SpyTag (Asp117)
(Asp117)
isisspontaneous.
spontaneous.

The
Theindustrial
industrialenzyme
enzyme glutaryl-7-aminocephalosporanic
glutaryl‐7‐aminocephalosporanicacid acidacylase
acylase(GA)
(GA)waswasfused
fused
with
with SpyTag. After overexpression and purification, the fusion enzyme SpyTag‐GA was
SpyTag. After overexpression and purification, the fusion enzyme SpyTag-GA was
loaded onto a SpyCatcher-derivatized carrier. A high immobilization efficiency
loaded onto a SpyCatcher‐derivatized carrier. A high immobilization efficiency (91%) and (91%) and
activity
activityrecovery
recovery(86%) were
(86%) achieved
were in ainvery
achieved shortshort
a very time compared
time comparedto the classical epoxy-
to the classical
mediated immobilization
epoxy‐mediated (immobilization
immobilization efficiency
(immobilization = 29%=and
efficiency 29%activity recovery
and activity = 3%)=
recovery
after 2 h of incubation. Using the same technology, the immobilization of SpyTag-GA
3%) after 2 h of incubation. Using the same technology, the immobilization of SpyTag‐GA was
attempted
was attempteddirectly from E.
directly coliE.
from cell lysates.
coli Also inAlso
cell lysates. this in
case,
thisthe SpyTag-GA
case, was immo-
the SpyTag‐GA was
bilized on the SpyCatcher-derivatized carrier in high yields (86%) and with high activity
immobilized on the SpyCatcher‐derivatized carrier in high yields (86%) and with high
recovery (91%) compared to the epoxy-mediated immobilization samples, for which the
activity recovery (91%) compared to the epoxy‐mediated immobilization samples, for
immobilization efficiency and activity recovery of SpyTag-GA were only 25% and 3%,
which the immobilization efficiency and activity recovery of SpyTag‐GA were only 25%
respectively [101]. In another example, D-allulose 3-epimerase (DAEase) from Clostrid-
and 3%, respectively [101]. In another example, D‐allulose 3‐epimerase (DAEase) from
ium cellulolyticum was successfully immobilized via covalent attachment on SpyCatcher-
Clostridium cellulolyticum was successfully immobilized via covalent attachment on
derivatized carrier through Spy chemistry and employed for the synthesis of D-allulose
SpyCatcher‐derivatized carrier through Spy chemistry and employed for the synthesis of
from D-fructose. The immobilized derivative maintained 80% of activity after seven con-
D‐allulose from D‐fructose. The immobilized derivative maintained 80% of activity after
secutive cycles and 25 days of storage [102]. Finally, peptide-N-glycosidase F (PNGase F)
seven consecutive cycles and 25 days of storage [102]. Finally, peptide‐N‐glycosidase F
fused to the SpyCatcher domain was site-specifically and covalently immobilized onto
(PNGase F) fused to the SpyCatcher domain was site‐specifically and covalently
magnetic nanoparticles derivatized with SpyTag. The final biocatalyst exhibited good
immobilized onto magnetic nanoparticles derivatized with SpyTag. The final biocatalyst
thermal stability and deglycosylation activity of different glycoproteins releasing various
exhibited good thermal stability and deglycosylation activity of different glycoproteins
types of glycans (high-mannose, sialylated, and hybrid). Furthermore, after five reaction
releasing various types of glycans (high‐mannose, sialylated, and hybrid). Furthermore,
cycles, the immobilized enzyme retained 78% of the initial activity [103].
after five reaction cycles, the immobilized enzyme retained 78% of the initial activity [103].
Enzyme-Mediated Immobilization Driven by Tag Sequences
Enzyme‐Mediated Immobilization Driven by Tag Sequences
In addition to the self-ligating systems, the covalent bond formation between the
In addition to the self‐ligating systems, the covalent bond formation between the
enzyme and the carrier can be mediated by an enzyme in vitro (enzyme-mediated immo-
enzyme and the carrier can be mediated by an enzyme in vitro (enzyme‐mediated
bilization). The transpeptidase Sortase A (SrtA) generally recognizes the LPXTG motif
immobilization). The transpeptidase Sortase A (SrtA) generally recognizes the LPXTG
(where X is any amino acid except cysteine). SrtA cleaves the amide bond between the
motif (where X is any amino acid except cysteine). SrtA cleaves the amide bond between
threonine and glycine in the LPXTG motif by forming a thioester intermediate, which can
the threonine and glycine in the LPXTG motif by forming a thioester intermediate, which
can then be displaced by an amine nucleophile, typically the free amino group of a
polyglycine substrate, forming a stable peptide bond. SrtA can be used as a sort of
molecular “stapler” that mediates site‐specific cross‐linking between proteins fused with
Molecules 2025, 30, 939 21 of 33

Molecules 2025, 30, x FOR PEER REVIEW

thenbe displaced by an amine nucleophile, typically the free amino group of a22polyg- of 35

lycine substrate, forming a stable peptide bond. SrtA can be used as a sort of molecular
“stapler” that mediates site-specific cross-linking between proteins fused with the LPXTG
the
motifLPXTG motif
at their at their and
C-terminus C‐terminus
an amine and an amine
group group (or motif)
(or glycinamide glycinamide
presentmotif)
on thepresent
carrier
on the carrier
(Scheme (Scheme
14) [104]. SrtA 14)
can [104]. SrtAproduced
be simply can be simply produced
in-house in E. coliin‐house in E. coli in
in large amounts, large
making
amounts, making this
this immobilization immobilization
technique technique cost‐effective.
cost-effective.

Scheme 14. Site-specific

Scheme 14. Site‐specific covalent
covalentenzyme
enzymeimmobilization
immobilizationmediated
mediated byby Sortase
Sortase A. Instead
A. Instead of
of pen-
pentaglycine,
taglycine, the the
solidsolid carrier
carrier can becan be derivatized
derivatized with substrate
with substrate nucleophiles
nucleophiles that consist
that consist of a
of a smaller
numbernumber
smaller of glycine
of residues or simpleoramine
glycine residues simplegroups.
amine Alternatively, the LPXTG
groups. Alternatively, therecognition sequence
LPXTG recognition
can be conjugated
sequence to the solid
can be conjugated carrier,
to the solidand the protein
carrier, and theofprotein
interest
ofcan be tagged
interest can bewith an N-terminal
tagged with an N‐
oligoglycine motif.
terminal oligoglycine motif.

Thermobifida fusca YX β-glucosidase (BGL) and Streptococcus bovis 148 α-amylase

Thermobifida fusca YX ‐glucosidase (BGL) and Streptococcus bovis 148 ‐amylase
(AmyA) were successfully immobilized via SrtA-mediated transpeptidation on polystyrene
(AmyA) were successfully immobilized via SrtA‐mediated transpeptidation on
nanoparticles derivatized with a tri-glycine tag. Both immobilized BGL and AmyA ex-
polystyrene nanoparticles derivatized with a tri‐glycine tag. Both immobilized BGL and
hibited a 3.0- or 1.5-fold, respectively, higher activity compared to the same enzymes
AmyA exhibited a 3.0‐ or 1.5‐fold, respectively, higher activity compared to the same
immobilized by classical (random) covalent immobilization. Moreover, the two biocatalysts
enzymes immobilized by classical (random) covalent immobilization. Moreover, the two
were able to retain more than 80% of initial activity after 10 consecutive reaction cycles,
biocatalysts were able to retain more than 80% of initial activity after 10 consecutive
suggesting good recyclability [105]. In another study, the use of graphene-oxide carriers
reaction cycles, suggesting good recyclability [105]. In another study, the use of graphene‐
functionalized simply with ethylenediamine as an amine donor group in sortase-mediated
oxide carriers functionalized simply with ethylenediamine as an amine donor group in
immobilization was demonstrated to be more effective than pentaglycine-derivatized
sortase‐mediated immobilization was demonstrated to be more effective than
graphene-oxide carriers. Silica-based carriers derivatized with pentaglycine were more
pentaglycine‐derivatized graphene‐oxide carriers. Silica‐based carriers derivatized with
effective than amine-functionalized silica [106]. These results suggested that the type of
pentaglycine were more effective than amine‐functionalized silica [106]. These results
amine nucleophile used for the derivatization of the carrier and the type of carrier have
suggested that the type of amine nucleophile used for the derivatization of the carrier and
to be carefully evaluated since they can affect the SrtA-mediated immobilization process,
the type of carrier have to be carefully evaluated since they can affect the SrtA‐mediated
thus altering the final properties of the immobilized derivative. Based on these results,
immobilization process, thus altering the final properties of the immobilized derivative.
Candida antarctica lipase B (CalB) was covalently immobilized on the surface of amine-
Based on these results, Candida antarctica lipase B (CalB) was covalently immobilized on
functionalized graphene oxide (GO) nanoparticles by SrtA-mediated immobilization [107].
the surface of amine‐functionalized graphene oxide (GO) nanoparticles by SrtA‐mediated
Almost 60% of CalB was immobilized on the amine-derivatized support from crude extract
immobilization [107]. Almost 60% of CalB was immobilized on the amine‐derivatized
after 16 h of incubation with an activity recovery of 132%, probably due to the uniform ori-
support from crude extract after 16 h of incubation with an activity recovery of 132%,
entation of the enzyme molecules on the carrier that facilitates the accessibility of substrates
probably due to the uniform orientation of the enzyme molecules on the carrier that
to the active sites. Moreover, the oriented immobilized derivative was employed in the
facilitates the accessibility of substrates to the active sites. Moreover, the oriented
hydrolysis of fish oil, showing a 2.5-fold higher selectivity in the release of cis-5, 8, 11, 14,
immobilized derivative was employed in the hydrolysis of fish oil, showing a 2.5‐fold
and 17-eicosapentaenoic acid compared to the immobilized derivative obtained by random
higher selectivity in the release of cis‐5, 8, 11, 14, and 17‐eicosapentaenoic acid compared
immobilization. By using this strategy, two unstable membrane-bound glycosyltransferases
to the immobilized derivative obtained by random immobilization. By using this strategy,
were immobilized on amino-derivatized carriers [108]. Immobilized recombinant human
two unstable membrane‐bound glycosyltransferases were immobilized on amino‐
derivatized carriers [108]. Immobilized recombinant human β1,4‐galactosyltranseferase
or recombinant Helicobacter pylori R1,3‐fucosyltransferase were used successfully in the
repetitive synthesis of Lewis X antigen.
Molecules 2025, 30, 939 22 of 33

β1,4-galactosyltranseferase or recombinant Helicobacter pylori R1,3-fucosyltransferase were

used successfully in the repetitive synthesis of Lewis X antigen.
Microbial transglutaminases (mTG) can be used alternatively to SrtA to mediate an ori-
ented and covalent immobilization of glutamine-tagged enzymes onto amine-derivatized
solid carriers [109]. Over the years, different highly reactive Q-tags for the Bacillus subtilis
mTG [110] and new mTG with high substrate specificity [111] were discovered, providing
new tools for mTG-mediated bioconjugation. The alkaline phosphatase (AP) from E. coli
tagged with a hexapeptide containing one glutamine residue (MLAQGS) was covalently
and site-specifically immobilized by transglutaminase mediated immobilization onto a
polystyrene surface physically coated with β-casein or bovine serum albumin displaying
reactive lysine residues [112] as well as onto aminated magnetic particles [113]. Immobi-
lization efficiency was affected by the type of amine group present on the surface magnetic
particles. The derivatization of the carrier with diethyleneglycol bis (3-aminopropyl)ether
(DGBE) allows the non-specific physical adsorption of the enzyme on carrier surface to
be reduced and, thus, only the site-specific covalent immobilized enzyme to be obtained.
The immobilized AP exhibited 93% of the initial activity after 10 rounds of recycling. The
biotin ligase BirA from E. coli, frequently used for in vitro biotinylation of recombinant
proteins, was immobilized onto amine-modified magnetic microsphere by mTG-promoted
condensation [114]. The site-specifically immobilized BirA exhibited approximately 95%
enzymatic activity of the soluble BirA, and no significant loss in activity after 10 rounds
of recycling was observed. In addition, the immobilized BirA could be easily recovered
from the solution via a simple magnetic separation. Thus, the immobilized BirA repre-
sents a robust biocatalyst of general use for efficient and economically feasible in vitro
biotinylations. An engineered enterokinase fused at the C-terminus with the MLAQGS
tag was site-specifically immobilized onto amine-modified magnetic nanoparticles via
mTG-catalyzed bioconjugation for the development of a reusable biocatalyst [115]. Upon
the site-specific immobilization, approximately 90% enterokinase enzymatic activity was
retained, and the biocatalyst exhibited more than 85% of initial enzymatic activity regard-
less of storage or reusable stability over a month. The developed biocatalyst was further
applied to remove the His-tag from the triplet of glucagon-like peptide-1 (GLP-1), showing
remarkable reusability without a significant decrease in enzymatic activity after 10 reaction
cycles. PNGase F was fused at the N-terminus with a WALQRPH tag that allowed a soluble
expression of the enzyme and its immobilization via mTG-catalysis onto amine-modified
magnetic particles [116]. The immobilized derivative exhibited the same N-deglycosylation
activity as its soluble counterpart, as well as excellent stability and good reusability
(79% residual activity after five reaction cycles), thus making it an efficient tool for
N-glycan analysis.

3.2.2. Sequence Engineering-Mediated Bio-Orthogonal Covalent Immobilization

Another strategy that allows greater versatility in enzyme orientation is the engineer-
ing of the sequence of the target enzyme by the introduction of natural amino acids or
unnatural amino acids in specific regions of the protein. In order to apply this strategy, the
three-dimensional structure of the enzyme (or some analogous one) should be resolved
in order to introduce these modifications in a rational way. Moreover, it has to be taken
into consideration that these modifications can interfere with the expression/stability of
the recombinant-engineered enzyme.

Incorporation of Naturally Reactive Occurring Amino Acids by Site-Specific Mutagenesis

Enzyme immobilization can be oriented by the incorporation of naturally reactive
occurring amino acids by site-specific mutagenesis into the biocatalyst sequence. Naturally,
Molecules 2025, 30, 939 23 of 33

amino acids generally exploited also for random immobilization protocols (e.g., Lys or
Cys) can be introduced on the protein surface by site-directed mutagenesis both as extra
residues localized just on a desired region of the protein surface or as unique residues.
The addition of extra Lys residues localized in a specific region of the protein surface
can allow an oriented immobilization protocol to be achieved by forcing the enzyme to
interact with the carrier, mostly with a small enriched Lys surface area. This strategy
generally requires the replacement of several amino acids present on a small surface area
of the protein, and at the same time, the deletion of other lysine residues localized in areas
that are not of interest could be necessary. However, introducing several mutations in a
small surface area can negatively affect protein stability. Thus, the effect of the substitution
is not easy to predict. The catalytic activity of the immobilized Penicillin G acylase (PGA)
from E. coli on aldehyde agarose was improved by designing different engineered protein
variants containing extra lysine residues on different regions of the protein since 72%
of the wild-type PGA was immobilized on the carrier mainly through the Lys residues
positioned on the same side of the active site, thus blocking the entrance to the bulky β-
lactams substrates. The variant 3xLys-PGA, possessing three extra Lys residues alternating
with three Gly residues at the end of the β chain, on the opposite site of the active site,
showed better accessibility of the substrates since 63% of its active site was facing the
reaction medium [117]. Following the same strategy, the immobilization yield of xylanase
from Chaetomium globosum onto SiO2 nanoparticles was improved up to 99% compared to
wild-type enzyme by designing a penta-mutated variant (Asn172Lys-His173Lys-Ser176Lys-
Lys133Ala-Lys148Ala). The wild-type enzyme showed low immobilization yield due to
a lack of lysine residues present on its surface. By applying a mutation energy-based in
silico screening approach and some rational biochemical evaluations of surface residues,
three residues were selected for lysine substitution: Asn172, His173, and Ser176, while
two naturally occurring lysins 133 and 148, which were located near the active site region
were mutated to alanine [118].
A single cysteine residue was introduced, using site-directed mutagenesis, into the
PGA at six different regions of the enzyme that were rich in Lys residues. The different vari-
ants were immobilized on a disulfide-containing carrier through covalent immobilization,
exhibiting excellent stability and selectivity in the synthesis of cephalosporines derivatives
through additional protein rigidification. The replacement of Gln380 with Cys produced an
immobilized derivative that was 30-fold more stable than the soluble preparation toward
heat and organic co-solvents and preserved 90% of the initial activity [119].

Incorporation of Unnatural Amino Acids

The incorporation of unnatural amino acids with unique reactive groups into the
enzyme sequence at a single site or multiple sites is an elegant strategy to achieve high
precision in covalently bound formation between an enzyme and a carrier. Genetic code
expansion can be used to insert a plethora of unnatural amino acids into the protein
sequence. Based on amino acid type, a variety of covalent ligation reactions can be applied.
Among all, copper-catalyzed azide–alkyne cycloadditions, strain-promoted azide–alkyne
cycloadditions, and inverse-electron-demand Diels–Alder and Glasser-Hay reactions were
applied for enzyme immobilization onto solid carriers (Scheme 15).
Molecules2025,
Molecules 2025,30,
30,939
x FOR PEER REVIEW 25 of 35
24 of 33

Scheme15.
Scheme 15.Site-specific
Site‐specific covalent
covalent immobilization
immobilization mediated
mediated by unnatural
by unnatural amino
amino acids. (A)acids. (A) Cu(I)‐
Cu(I)-catalyzed
azide–alkyne cycloaddition (CuAAC)
catalyzed azide–alkyne reaction,
cycloaddition (B) strain-promoted
(CuAAC) azide–alkyne
reaction, (B) cycloaddition
strain‐promoted (SPACC)
azide–alkyne
reaction, (C) inverse electron-demand Diels–Alder (IEDDA) reaction, (D) Glaser–Hay reaction.
cycloaddition (SPACC) reaction, (C) inverse electron‐demand Diels–Alder (IEDDA) reaction, (D)
Glaser–Hay reaction.
Molecules 2025, 30, 939 25 of 33

Copper-Catalyzed Azide–Alkyne Cycloadditions (CuAAC)

The copper-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes is one
of the best-known click reactions (Scheme 15A). Several Cu(I) sources can be used to cat-
alyze the reaction, but preferably, the catalyst is prepared in situ by the reduction of Cu(II)
salts [120]. This reaction is usually performed in aqueous buffer to produce a stable and in-
ert 1,4-triazole. Different variants of T4 lysozyme containing p-propargyloxyphenylalanine
(pPa) at different positions (Thr21, Lys35, Asn53, Leu91, Lys135, and Lys162) were produced
by amber stop codon strategy and immobilized on superparamagnetic beads [121]. The ac-
tivity of the immobilized enzyme was shown to vary depending on the site of conjugation:
three variants in which the insertion of unnatural amino acid pPa were introduced far from
the active site, Asn53pPa, Lys135pPa, and Lys162pPa, maintained high activity (70–85%)
while the other two variants, Thr21pPa and Lys35pPa, showed a dramatic decrease in activ-
ity (<10% for Thr21pPa variant). The Leu91pPa variant had the highest retained activity of
all the after immobilization and was further investigated. The site-specific immobilization
of this variant enhanced its stability toward freeze-thaw cycles (80% retained activity) and
the denaturant urea when compared to the enzyme immobilized in a random manner on
epoxy-modified beads as well as soluble lysozyme.
The use of a copper catalyst increases the azide-alkyne click reaction rate, but the
formation of reactive oxygen species generated during these reactions has been well docu-
mented to negatively affect the structure and functional activity of proteins.

Strain-Promoted Azide-Alkyne Cycloadditions (SPAAC)

Strain-promoted azide-alkyne click reaction is a metal-free [3+2] cycloaddition reaction
widespread used for enzyme immobilization. Cyclooctyne is the smallest stable cyclic
alkyne with a 17◦ distortion from the preferred linear alkyne geometry, thus allowing a
sufficient ring strain able to promote a metal-free cycloaddition of azides (Scheme 15B).
This reaction generally proceeds at a slow rate compared to the copper-catalyzed reaction
and generally requires high protein concentrations and overnight incubations. These
conditions can promote nonspecific protein adsorption, protein aggregation, and loss of
enzyme activity.
Amber codon suppression was used to replace five tyrosine residues (50, 137, 243,
274, and 355) with 4-azido-L-phenylalanine in Geobacillus sp. SBS-4S lipase [122]. The
variants were conjugated to mesocellular siliceous foams derivatized with cyclooctene
groups using the SPAAC reaction. The variant AzPhe-Lip243 showed higher catalytic
activity and higher thermostability compared to the wild-type enzyme conventionally
immobilized by using glutaraldehyde. In another example, 4-azido-L-phenylalanine, was
incorporated into five specific sites of a laccase from Streptomyces coelicolor (SLAC) and
immobilized onto multi-walled carbon nanotube electrode derivatized with the bifunctional
linker cyclooctynyloxyethyl 1-pyrenebutyrate [123]. A remarkably high direct electron
transfer efficiency and a stable current that deteriorated only around 14% following 8 days
of solution-phase incubation at ambient temperature were achieved with the electrode
containing the click-incorporated Glu47AzF variant of SLAC.

Inverse-Electron-Demand Diels-Alder Reactions (IEDDA)

The inverse-electron-demand Diels-Alder (IEDDA) reaction involves the cycloaddition
of an electron-rich dienophile with an electron-poor diene (Scheme 15C). Cycloaddition
of tetrazines with strained trans-cyclooctenes does not require a catalyst, shows a higher
tunable reaction rate compared to 3+2 cycloadditions, and can be performed in different
buffers, showing high biocompatibility. Human carbonic anhydrase II was genetically engi-
neered with a tetrazine containing unnatural amino acid (Tet2.0) at three different positions
Molecules 2025, 30, 939 26 of 33

(186, 233, 20) and immobilized via IEDDA onto trans-cyclooctene modified surfaces [124].
This immobilization strategy allowed the enzyme load to be controlled by modulating
the amount of protein introduced into the system also at low protein concentration (nM).
Moreover, the short reaction time (minutes) required for the immobilization procedure
minimizes protein denaturation and nonspecific adsorption, allowing immobilized surfaces
to be prepared with exceptional levels of homogeneity.

Glaser-Hay
The Glaser-Hay bioconjugation between terminal alkenes allows a highly stable and
rigid linear C-C bond to be obtained under mild conditions (Scheme 15D) [125]. The
hyperthermophilic carboxylesterase P1 from Sulfolobus solfataricus (SSo EST1) was immo-
bilized onto an epoxy-activated Sepharose resin derivatized with propargyl alcohol via
Glaser–Hay reaction [126]. Four tyrosine residues were mutated into the tyrosine derivative
p-propargyloxyphenylalanine (pPrF) (Tyr90 Tyr116, Tyr191, and Tyr214). Incorporation
of pPrF at sites 90 and 116 resulted in similar or slightly lower protein expression yields
than wild-type; however, incorporation at residues 191 and 214 resulted in significantly
decreased yields. The SSo EST1-116 mutant maintained comparable activity to the wild-
type protein, suggesting minimal perturbations caused by pPrF, whereas the SSo EST1-214
mutant shows significantly reduced activity. The immobilized enzyme exhibited high
performance in organic solvents, recyclability, and stability at room temperature for over
2 years.

4. Evaluation of Immobilization Outcome

Immobilization, considered the attachment of an enzyme on or into a porous solid
carrier, is a relatively simple process. However, many factors can affect the expressed
activity of the final enzyme derivative: enzyme distortion, mass transfer limitations, steric
hindrance, enzyme leaching, carrier with low loading capacity, physical features of the
carrier, etc. All these maters should be considered at the end of the immobilization proce-
dure in order to fully understand the effects of the immobilization on enzyme activity. The
three parameters that should be provided to evaluate the success of enzyme immobi-
lization are the immobilization yield, the immobilization efficiency, and the activity re-
covery. These parameters allow the enzyme activity to be evaluated during the whole
immobilization process.
The immobilization yield is used to describe the percentage of enzyme activity that
has been immobilized onto the carrier. This parameter is calculated by dividing the residual
activity found in the supernatant at the end of the immobilization procedure by the activity
found initially in the supernatant before the addition of the solid carrier.

Immobilization yield (%) = (Unitst0 − Unitsendpoint )/(Unitst0 ) × 100

The immobilized activity at the endpoint is determined by measuring the total residual
enzyme activity that remains in the supernatant at the endpoint of the immobilization
and by subtracting this activity from the total initial activity. In order to be sure that the
decrease of activity observed in the supernatant at the end of the immobilization is due
to the effective binding of the enzyme to the carrier, a parallel stability experiment should
be carried out in order to evaluate the possible deactivation of the soluble enzyme in the
immobilization conditions. The evaluation of protein concentration during the immobiliza-
tion can also be used to determine the immobilization yield. However, the disappearance
of the protein from the supernatant sometimes could be misleading, especially when a
crude protein mixture is used because the different proteins present in the mixture can
have different immobilization yields. Generally, both protein concentration and enzyme
Molecules 2025, 30, 939 27 of 33

activity have to be monitored during an immobilization process in order to be able to rule

out any deactivation of the soluble enzyme and to determine the protein loading of the
immobilized biocatalyst [1,19].
The immobilization efficiency describes the percentage of enzyme activity that was
bound into the final derivative. These parameters take into account the activity of the final
immobilized derivative and the immobilization yield [1,19].

Efficiency (%) = (observed activity (U/g))/(immobilized activity (U/g)) × 100

Finally, the activity recovery describes the overall success of an immobilization proto-
col. It correlates the initially loaded activity and the final activity observed in the immobi-
lized biocatalyst [1,19].

Activity recovery (%) = (observed activity (U/g))/(loaded activity (U/g)) × 100

These parameters should be reported in all papers dealing with enzyme immobilization
in order to allow the reader to fully understand what is going on during the immobilization
process as well as the properties of the final immobilized derivative obtained.

5. Conclusions
Protein immobilization represents a versatile and promising solution to address the
challenges associated with enzyme applications in industrial processes.
This comprehensive review highlights the numerous advantages offered by im-
mobilization techniques, including enhanced enzyme stability, simplified recovery and
reuse, and significant reductions in downstream processing and operational costs. These
benefits make immobilization a cornerstone for developing sustainable and efficient
biocatalytic processes.
Classical non-specific immobilization methods, such as adsorption, ionic binding, and
entrapment, are widely utilized due to their simplicity, cost-effectiveness, and high enzyme
loading capacities. However, these approaches often lack precise control over enzyme
orientation, which can negatively impact catalytic efficiency and stability. Covalent immo-
bilization methods provide stronger enzyme-support interactions and improved stability,
but they require careful optimization to minimize activity loss caused by conformational
changes during the process.
Oriented immobilization methods, leveraging advanced protein engineering tech-
niques, represent a significant step forward in maximizing enzyme activity. By introducing
specific functional tags or unique amino acid residues, these methods ensure the optimal
orientation of enzymes on supports, improving substrate accessibility and catalytic effi-
ciency. Such approaches are particularly valuable for applications where enzyme activity
and selectivity are critical, such as pharmaceutical synthesis or fine chemical production.
Despite the clear advantages, challenges remain. Immobilization can lead to mass
transfer limitations, reduced activity with insoluble substrates, or enzyme denaturation if
protocols are not carefully designed. Hybrid approaches that combine protein engineering
and immobilization offer a promising solution to overcome these limitations, enabling the
development of tailored biocatalysts that exhibit both high stability and activity.

Author Contributions: M.S.R. and T.B. have equally contributed to the conceptualization,
writing—original draft preparation, and writing—review and editing of the manuscript. All authors
have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Molecules 2025, 30, 939 28 of 33

Informed Consent Statement: Not applicable.

Data Availability Statement: No new data were created or analyzed in this study.

Acknowledgments: This paper is part of the project NODES, which has received funding from the
MUR—M4C2 1.5 of PNRR funded by the European Union—NextGenerationEU (Grant agreement no.
ECS00000036).

Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Boudrant, J.; Woodley, J.M.; Fernandez-Lafuente, R. Parameters Necessary to Define an Immobilized Enzyme Preparation. Process
Biochem. 2020, 90, 66–80. [CrossRef]
2. Sheldon, R.A.; Brady, D. Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis. ChemSusChem 2019, 12, 2859–2881.
[CrossRef] [PubMed]
3. O’Connell, A.; Barry, A.; Burke, A.J.; Hutton, A.E.; Bell, E.L.; Green, A.P.; O’Reilly, E. Biocatalysis: Landmark Discoveries and
Applications in Chemical Synthesis. Chem. Soc. Rev. 2024, 53, 2828. [CrossRef]
4. Rossino, G.; Robescu, M.S.; Licastro, E.; Tedesco, C.; Martello, I.; Maffei, L.; Vincenti, G.; Bavaro, T.; Collina, S. Biocatalysis: A
Smart and Green Tool for the Preparation of Chiral Drugs. Chirality 2022, 34, 1403–1418. [CrossRef] [PubMed]
5. Wu, S.; Snajdrova, R.; Moore, J.C.; Baldenius, K.; Bornscheuer, U.T. Biocatalysis: Enzymatic Synthesis for Industrial Applications.
Angew. Chem. Int. Ed. 2021, 60, 88–119. [CrossRef]
6. Yi, D.; Bayer, T.; Badenhorst, C.P.S.; Wu, S.; Doerr, M.; Hohne, M.; Bornscheuer, U.T. Recent Trends in Biocatalysis. Chem. Soc. Rev.
2021, 50, 8003. [CrossRef] [PubMed]
7. Bié, J.; Sepodes, B.; Fernandes, P.C.B.; Ribeiro, M.H.L. Enzyme Immobilization and Co-Immobilization: Main Framework,
Advances and Some Applications. Processes 2022, 10, 494. [CrossRef]
8. Basso, A.; Serban, S. Industrial Applications of Immobilized Enzymes—A Review. Mol. Catal. 2019, 479, 110607. [CrossRef]
9. Sheldon, R.A.; Woodley, J.M. Role of Biocatalysis in Sustainable Chemistry. Chem. Rev. 2018, 118, 801–838. [CrossRef] [PubMed]
10. Romero-Fernandez, M.; Paradisi, F. General Overview on Immobilization Techniques of Enzymes for Biocatalysis. In Catalysts
Immobilization; John Wiley & Sons, Ltd.: New York, NY, USA, 2019; pp. 409–435.
11. Bilal, M.; Iqbal, H.M.N. Tailoring Multipurpose Biocatalysts via Protein Engineering Approaches: A Review. Catal. Lett. 2019, 149,
2204–2217. [CrossRef]
12. Bernal, C.; Rodriguez, K.; Martinez, R. Integrating Enzyme Immobilization and Protein Engineering: An Alternative Path for the
Development of Novel and Improved Industrial Biocatalysts. Biotechnol. Adv. 2018, 36, 1470–1480. [CrossRef]
13. Redeker, E.S.; Ta, D.T.; Cortens, D.; Billen, B.; Guedens, W.; Adriaensens, P. Protein Engineering for Directed Immobilization.
Bioconjug. Chem. 2013, 24, 1761–1777. [CrossRef] [PubMed]
14. Guisan, J.M. Immobilization of Enzymes and Cells: Methods and Protocols, Methods in Molecular Biology; Springer Science Business
Media, LLC., part of Springer Nature: Berlin/Heidelberg, Germany, 2020.
15. Bolivar, J.M.; Woodley, J.M.; Fernandez-Lafuente, R. Is Enzyme Immobilization a Mature Discipline? Some Critical Considerations
to Capitalize on the Benefits of Immobilization. Chem. Soc. Rev. 2022, 51, 6251–6290. [CrossRef]
16. Sheldon, R.A.; Basso, A.; Brady, D. New Frontiers in Enzyme Immobilisation: Robust Biocatalysts for a Circular Bio-Based
Economy. Chem. Soc. Rev. 2021, 50, 5850–5862. [CrossRef] [PubMed]
17. Sheldon, R.A. Enzyme Immobilization: The Quest for Optimum Performance. Adv. Synth. Catal. 2007, 349, 1289–1307. [CrossRef]
18. Cantone, S.; Ferrario, V.; Corici, L.; Ebert, C.; Fattor, D.; Spizzo, P.; Gardossi, L. Efficient Immobilisation of Industrial Biocatalysts:
Criteria and Constraints for the Selection of Organic Polymeric Carriers and Immobilisation Methods. Chem. Soc. Rev. 2013, 42,
6262–6276. [CrossRef]
19. Sheldon, R.A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42, 6223–6235.
[CrossRef]
20. Zucca, P.; Fernandez-Lafuente, R.; Sanjust, E. Agarose and Its Derivatives as Supports for Enzyme Immobilization. Molecules
2016, 21, 1577. [CrossRef] [PubMed]
21. Maghraby, Y.R.; El-Shabasy, R.M.; Ibrahim, A.H.; Azzazy, H.M.E.-S. Enzyme Immobilization Technologies and Industrial
Applications. ACS Omega 2023, 8, 5184–5196. [CrossRef]
22. Kumari, A.; Kaur, B.; Srivastava, R.; Sangwan, R.S. Isolation and Immobilization of Alkaline Protease on Mesoporous Silica and
Mesoporous ZSM-5 Zeolite Materials for Improved Catalytic Properties. Biochem. Biophys. Rep. 2015, 2, 108–114. [CrossRef]
[PubMed]
23. Sondhi, S.; Kaur, R.; Kaur, S.; Kaur, P.S. Immobilization of Laccase-ABTS System for the Development of a Continuous Flow
Packed Bed Bioreactor for Decolorization of Textile Effluent. Int. J. Biol. Macromol. 2018, 117, 1093–1100. [CrossRef] [PubMed]
Molecules 2025, 30, 939 29 of 33

24. Nguyen, H.H.; Kim, M. An Overview of Techniques in Enzyme Immobilization. Appl. Sci. Converg. Technol. 2017, 26, 157–163.
[CrossRef]
25. Luo, J.; Wan, Y.; Song, S.; Zhang, H.; Zhang, H.; Zhang, J.; Wan, Y. Biocatalytic Membrane: Go Far beyond Enzyme Immobilization.
Eng. Life Sci. 2020, 20, 441–450. [CrossRef] [PubMed]
26. Cen, Y.-K.; Liu, Y.-X.; Xue, Y.-P.; Zheng, Y.-G. Immobilization of Enzymes in/on Membranes and Their Applications. Adv. Synth.
Catal. 2019, 361, 5500–5515. [CrossRef]
27. Brena, B.; Gonzalez-Pombo, P.; Batista-Viera, F. Immobilization of Enzymes: A Literature Survey. In Immobilization of Enzymes and
Cells; Methods in Molecular Biology; Guisan, J., Ed.; Humana Press: Totowa, NJ, USA, 2013; Volume 1051.
28. Pantić, N.; Prodanović, R.; Ðurd̄ić, K.I.; Polović, N.; Spasojević, M.; Prodanović, O. Optimization of Phenol Removal with
Horseradish Peroxidase Encapsulated within Tyramine-Alginate Micro-Beads. Environ. Technol. Innov. 2021, 21, 101211.
[CrossRef]
29. Guan, T.; Liu, B.; Wang, R.; Huang, Y.; Luo, J.; Li, Y. The Enhanced Fatty Acids Flavor Release for Low-Fat Cheeses by Carrier
Immobilized Lipases on O/W Pickering Emulsions. Food Hydrocoll. 2021, 116, 106651. [CrossRef]
30. Rother, C.; Nidetzky, B. Enzyme Immobilization by Microencapsulation: Methods, Materials, and Technological Applications.
Encycl. Ind. Biotechnol. 2014, 7, 1–21. [CrossRef]
31. Ortiz, C.; Ferreira, M.L.; Barbosa, O.; Rodrigues, R.C.; Berenguer-Murcia, Á.; Briand, L.E.; Fernandez-Lafuente, R. Novozym 435:
The “Perfect” Lipase Immobilized Biocatalyst? Catal. Sci. Technol. 2019, 9, 2380. [CrossRef]
32. Rodrigues, R.C.; Virgen-Ortiz, J.J.; dos Santos, J.C.S.; Berenguer-Murcia, Á.; Alcantara, A.R.; Barbosa, O.; Ortiz, C.; Fernandez-
Lafuente, R. Immobilization of Lipases on Hydrophobic Supports: Immobilization Mechanism, Advantages, Problems, and
Solutions. Biotechnol. Adv. 2019, 37, 746–770. [CrossRef]
33. Semproli, R.; Robescu, M.S.; Sangiorgio, S.; Pargoletti, E.; Bavaro, T.; Rabuffetti, M.; Cappelletti, G.; Speranza, G.; Ubiali, D. From
Lactose to Alkyl Galactoside Fatty Acid Esters as Non-ionic Biosurfactants: A Two-step Enzymatic Approach to Cheese Whey
Valorization. ChemPlusChem 2023, 88, e202200331. [CrossRef]
34. Kumar, R.; Maikhuri, V.K.; Mathur, D.; Kumar, M.; Singh, N.; Prasad, A.K. Novozyme-435: Perfect Catalyst for Chemo- and
Regio-Selective Synthesis of Modified Carbohydrates—A Review. Biocatal. Biotransform. 2023, 42, 19–33. [CrossRef]
35. Hoyos, P.; Perona, A.; Bavaro, T.; Berini, F.; Marinelli, F.; Terreni, M.; Hernáiz, M.J. Biocatalyzed Synthesis of Glycostructures with
Anti-Infective Activity. Acc. Chem. Res. 2022, 55, 2409–2424. [CrossRef] [PubMed]
36. Tanzi, L.; Robescu, M.S.; Marzatico, S.; Recca, T.; Zhang, Y.; Terreni, M.; Bavaro, T. Developing a Library of Mannose-Based
Mono- and Disaccharides: A General Chemoenzymatic Approach to Monohydroxylated Building Blocks. Molecules 2020, 25, 5764.
[CrossRef] [PubMed]
37. Li, Z.; Bavaro, T.; Tengattini, S.; Bernardini, R.; Mattei, M.; Annunziata, F.; Cole, R.B.; Zheng, C.; Sollogoub, M.; Tamborini, L.;
et al. Chemoenzymatic Synthesis of Arabinomannan (AM) Glycoconjugates as Potential Vaccines for Tuberculosis. Eur. J. Med.
Chem. 2020, 204, 112578–112589. [CrossRef] [PubMed]
38. Robescu, M.S.; Ubiali, D.; Semproli, R.; Bavaro, T. Stepping into the Biocatalysis Lab for Undergraduate Students: From Enzyme
Immobilization to Product Isolation. J. Chem. Educ. 2024, 101, 2790–2795. [CrossRef]
39. Truppo, M.D.; Strotman, H.; Hughes, G. Development of an Immobilized Transaminase Capable of Operating in Organic Solvent.
ChemCatChem 2012, 4, 1071–1074. [CrossRef]
40. Antecka, K.; Zdarta, J.; Siwińska-Stefańska, K.; Sztuk, G.; Jankowska, E.; Oleskowicz-Popiel, P.; Jesionowski, T. Synergistic
Degradation of Dye Wastewaters Using Binary or Ternary Oxide Systems with Immobilized Laccase. Catalysts 2018, 8, 402.
[CrossRef]
41. Bruni, M.; Robescu, M.S.; Ubiali, D.; Marrubini, G.; Vanna, R.; Morasso, C.; Benucci, I.; Speranza, G.; Bavaro, T. Immobilization
of g-glutamyl Transpeptidase from Equine Kidney for the Synthesis of Kokumi Compounds. ChemCatChem 2020, 12, 210–218.
[CrossRef]
42. Liese, A.; Seelbach, K.; Wandrey, C. (Eds.) Industrial Biotransformations; John Wiley & Sons: Hoboken, NJ, USA, 2006.
43. Rocchietti, S.; Ubiali, D.; Terreni, M.; Albertini, A.M.; Fernández-Lafuente, R.; Guisán, J.M.; Pregnolato, M. Immobilization and
Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis. Biomacromolecules
2004, 5, 2195–2200. [CrossRef] [PubMed]
44. Sheldon, R.A. Characteristic Features and Biotechnological Applications of Cross-Linked Enzyme Aggregates (CLEAs). Appl.
Microbiol. Biotechnol. 2011, 92, 467–477. [CrossRef]
45. Hanefeld, U.; Gardossi, L.; Magner, E. Understanding Enzyme Immobilisation. Chem. Soc. Rev. 2009, 38, 453–468. [CrossRef]
[PubMed]
46. Barbosa, O.; Ortiz, C.; Berenguer-Murcia, Á.; Torres, R.; Rodrigues, R.C.; Fernandez-Lafuente, R. Glutaraldehyde in Bio-Catalysts
Design: A Useful Crosslinker and a Versatile Tool in Enzyme Immobilization. RSC Adv. 2014, 4, 1583–1600. [CrossRef]
Molecules 2025, 30, 939 30 of 33

47. Danielli, C.; van Langen, L.; Boes, D.; Asaro, F.; Anselmi, S.; Provenza, F.; Renzi, M.; Gardossi, L. 2,5-Furandicarboxaldehyde
as a Bio-Based Crosslinking Agent Replacing Glutaraldehyde for Covalent Enzyme Immobilization. RSC Adv. 2022, 12, 35676.
[CrossRef] [PubMed]
48. Orrego, A.H.; Romero-Fern, M.; Millan-Linares, M.d.C.; del Mar Yust, M.; Guisan, J.M.; Rocha-Martin, J. Stabilization of Enzymes
by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent. Catalysts 2018,
8, 333. [CrossRef]
49. Guisan, J.M. Aldehyde-Agarose Gels as Activated Supports for Immobilization-Stabilization of Enzymes. Enzyme Microb. Technol.
1988, 10, 375–382. [CrossRef]
50. Ho, J.; Al-Deen, F.M.N.; Al-Abboodi, A.; Selomulya, C.; Xiang, S.D.; Plebanski, M.; Forde, G.M. N,N ′ -Carbonyldiimidazole-
Mediated Functionalization of Superparamagnetic Nanoparticles as Vaccine Carrier. Colloids Surf. B Biointerfaces 2011, 83, 83–90.
[CrossRef]
51. Mateo, C.; Palomo, J.M.; Fuentes, M.; Betancor, L.; Grazu, V.; López-Gallego, F.; Pessela, B.C.C.; Hidalgo, A.; Fernández-Lorente,
G.; Fernández-Lafuente, R.; et al. Glyoxyl Agarose: A Fully Inert and Hydrophilic Support for Immobilization and High
Stabilization of Proteins. Enzyme Microb. Technol. 2006, 39, 274–280. [CrossRef]
52. Rueda, N.; dos Santos, J.C.S.; Torres, R.; Ortiz, C.; Barbosa, O.; Fernandez-Lafuente, R. Improved Performance of Lipases
Immobilized on Heterofunctional Octyl-Glyoxyl Agarose Beads. RSC Adv. 2015, 5, 11212–11222. [CrossRef]
53. Palomo, J.M.; Filice, M.; Fernandez-Lafuente, R.; Terreni, M.; Guisan, J.M. Regioselective Hydrolysis of Different Peracetylated
B-monosaccharides by Immobilized Lipases from Different Sources. Key Role of the Immobilization. Adv. Synth. Catal. 2007, 349,
1969–1976. [CrossRef]
54. Babaki, M.; Yousefi, M.; Habibi, Z.; Brask, J.; Mohammadi, M. Preparation of Highly Reusable Biocatalysts by Immobilization of
Lipases on Epoxy-Functionalized Silica for Production of Biodiesel from Canola Oil. Biochem. Eng. J. 2015, 101, 23–31. [CrossRef]
55. Semproli, R.; Vaccaro, G.; Ferrandi, E.E.; Vanoni, M.; Bavaro, T.; Marrubini, G.; Annunziata, F.; Conti, P.; Speranza, G.; Monti,
D.; et al. Use of Immobilized Amine Transaminase from Vibrio fluvialis under Flow Conditions for the Synthesis of (S)-1-(5-
fluoropyrimidin-2-yl)-ethanamine. ChemCatChem 2020, 12, 1359–1367. [CrossRef]
56. Mallin, H.; Muschiol, J.; Bystrom, E.; Bornscheuer, U.T. Efficient Biocatalysis with Immobilized Enzymes or Encapsulated Whole
Cell Microorganism by Using the SpinChem Reactor System. ChemCatChem 2013, 5, 3529–3532. [CrossRef]
57. Crotti, M.; Robescu, M.S.; Bolivar, J.M.; Ubiali, D.; Wilson, L.; Contente, M.L. What’s New in Flow Biocatalysis? A Snapshot of
2020–2022. Front. Catal. 2023, 3, 1154452. [CrossRef]
58. Robescu, M.S.; Annunziata, F.; Somma, V.; Calvio, C.; Morelli, C.F.; Speranza, G.; Tamborini, L.; Ubiali, D.; Pinto, A.; Bavaro, T.
From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis
of Biologically Active Peptide Derivatives. J. Agric. Food Chem. 2022, 70, 13692–13699. [CrossRef] [PubMed]
59. dos Santos, J.C.S.; Rueda, N.; Barbosa, O.; Fernandez-Sanchez, J.F.; Medina-Castillo, A.L.; Ramon-Marquez, T.; Arias-Martos,
M.C.; Millan-Linares, C.; Pedroche, J.; del Mar Yust, M.; et al. Characterization of Supports Activated with Divinyl Sulfone as a
Tool to Immobilize and Stabilize Enzymes via Multipoint Covalent Attachment. Application to Chymotrypsin. RSC Adv. 2015,
5, 20639. [CrossRef]
60. Benítez-Mateos, A.I.; Sebastian, E.S.; Ríos-Lombardía, N.; Morís, F.; Gonzµlez-Sabín, J.; López-Gallego, F. Asymmetric Reduction
of Prochiral Ketones by Using Self-sufficient Heterogeneous Biocatalysts Based on NADPH-dependent Ketoreductases. Chem.
Eur. J. 2017, 23, 16843–16852. [CrossRef]
61. Stalder, K.; Benítez-Mateos, A.I.; Paradisi, F. Biocatalytic Synthesis of L-pipecolic Acid by a Lysine Cyclodeaminase: Batch and
Flow Reactors. ChemCatChem 2024, 16, e202301671. [CrossRef]
62. Kohn, J.; Wilchek, M. A New Approach (Cyano-Transfer) for Cyanogen Bromide Activation of Sepharose at Neutral pH, Which
Yields Activated Resins Free of Interfering Nitrogen Derivatives. Biochem. Biophys. Res. Commun. 1982, 107, 878–884. [CrossRef]
[PubMed]
63. Bavaro, T.; Tengattini, S.; Rezwan, R.; Chiesa, E.; Temporini, C.; Dorati, R.; Massolini, G.; Conti, B.; Ubiali, D.; Terreni, M. Design
of Epidermal Growth Factor Immobilization on 3D Biocompatible Scaffolds to Promote Tissue Repair and Regeneration. Sci. Rep.
2021, 11, 2629. [CrossRef] [PubMed]
64. Piazza, G.J.; Medina, M.B. Covalent Immobilization of Enzymes Using Commercially Available CDI-Activated Agarose. In
Immobilization of Enzymes and Cells; Methods in Biotechnology; Bickerstaff, G.F., Ed.; Humana Press: Totowa, NJ, USA, 1997;
Volume 1.
65. Hernandez, K.; Fernandez-Lafuente, R. Control of Protein Immobilization: Coupling Immobilization and Site-Directed Mutagen-
esis to Improve Biocatalyst or Biosensor Performance. Enzyme Microb. Technol. 2011, 48, 107–122. [CrossRef] [PubMed]
66. Wang, X.; Xia, N.; Liu, L. Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application
in Sensing. Int. J. Mol. Sci. 2013, 14, 20890–20912. [CrossRef] [PubMed]
Molecules 2025, 30, 939 31 of 33

67. Abad, M.; Velez, M.; Santamaria, C.; Guisan, J.M.; Matheus, P.R.; Vazquez, L.; Gazaryan, I.; Gorton, L.; Gibson, T.; Fernandez, V.M.
Immobilization of Peroxidase Glycoprotein on Gold Electrodes Modified with Mixed Epoxy-Boronic Acid Monolayers. J. Am.
Chem. Soc. 2002, 124, 12845–12853. [CrossRef] [PubMed]
68. Gutarra, M.L.E.; Mateo, C.; Freire, D.M.G.; Torres, F.A.G.; Castro, A.M.; Guisan, J.M.; Palomo, J.M. Oriented Irreversible
Immobilization of a Glycosylated Candida antarctica B Lipase on Heterofunctional Organoborane-Aldehyde Support. Catal. Sci.
Technol. 2011, 1, 260–266. [CrossRef]
69. Nazemi, S.A.; Olesinska, M.; Pezzella, C.; Varriale, S.; Lin, C.-W.; Corvini, P.F.-X.; Shahgaldian, P. Immobilisation and Stabilisation
of Glycosylated Enzymes on Boronic Acid-Functionalised Silica Nanoparticles. Chem. Commun. 2021, 57, 11960–11963. [CrossRef]
70. Rao, S.V.; Anderson, K.W.; Bachas, L.G. Oriented Immobilization of Proteins. Mikrochim. Acta 1998, 128, 127–143. [CrossRef]
71. Gupta, P.; Maqbool, T.; Saleemuddin, M. Oriented Immobilization of Stem Bromelain via the Lone Histidine on a Metal Affinity
Support. J. Mol. Catal. B Enzym. 2007, 45, 78–83. [CrossRef]
72. Freitas, A.I.; Domingues, L.; Aguiar, T.Q. Tag-Mediated Single-Step Purification and Immobilization of Recombinant Proteins
toward Protein-Engineered Advanced Materials. J. Adv. Res. 2022, 36, 249–264. [CrossRef]
73. Sunbul, M.; Yin, J. Site Specific Protein Labeling by Enzymatic Posttranslational Modification. Org. Biomol. Chem. 2009, 7,
3361–3371. [CrossRef]
74. Young, T.S.; Schultz, P.C. Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon. J. Biol. Chem. 2010, 285,
11039–11044. [CrossRef]
75. Pellis, A.; Vastano, M.; Quartinello, F.; Acero, E.H.; Guebitz, G.M. His-tag Immobilization of Cutinase 1 from Thermobifida
cellulosilytica for Solvent-free Synthesis of Polyesters. Biotechnol. J. 2017, 12, 1700322. [CrossRef] [PubMed]
76. Thompson, M.P.; Derrington, S.R.; Heath, R.S.; Porter, J.L.; Mangas-Sanchez, J.; Devine, P.N.; Truppo, M.D.; Turner, N.J. A Generic
Platform for the Immobilisation of Engineered Biocatalysts. Tetrahedron 2019, 75, 327–334. [CrossRef]
77. Böhmer, W.; Knaus, T.; Volkov, A.; Slot, T.K.; Shiju, N.R.; Cassimjee, K.E.; Mutti, F.G. Highly Efficient Production of Chiral
Amines in Batch and Continuous Flow by Immobilized ω-Transaminases on Controlled Porosity Glass Metal-Ion Affinity Carrier.
J. Biotechnol. 2019, 291, 52–60. [CrossRef]
78. Tentori, F.; Bavaro, T.; Brenna, E.; Colombo, D.; Monti, D.; Semproli, R.; Ubiali, D. Immobilization of Old Yellow Enzymes via
Covalent or Coordination Bonds. Catalysts 2020, 10, 260. [CrossRef]
79. de Andrade, B.C.; Gennari, A.; Renard, G.; Nervis, B.D.R.; Benvenutti, E.V.; Costa, T.M.H.; Nicolodi, S.; Pesce da Silveira, N.;
Chies, J.M.; Volpato, G.; et al. Synthesis of Magnetic Nanoparticles Functionalized with Histidine and Nickel to Immobilize
His-Tagged Enzymes Using β-Galactosidase as a Model. Int. J. Biol. Macromol. 2021, 184, 159–169. [CrossRef]
80. Robescu, M.S.; Tengattini, S.; Rabuffetti, M.; Speranza, G.; Terreni, M.; Bavaro, T. β-Mannosidase from Cellulomonas fimi:
Immobilization Study and Application in the β-Mannoside Synthesis. Catalysts 2023, 13, 1399. [CrossRef]
81. Cassimjee, K.E.; Kadow, M.; Wikmark, Y.; Svedendahl Humble, M.; Rothstein, M.L.; Rothstein, D.M.; Backvall, J.-E. A General
Protein Purification and Immobilization Method on Controlled Porosity Glass: Biocatalytic Applications. Chem. Commun. 2014,
50, 9134. [CrossRef] [PubMed]
82. Zeballos, N.; Comino, N.; Andrés-Sanz, D.; Santiago-Arcos, J.; Azkargorta, M.; Elortza, F.; Diamanti, E.; López-Gallego, F.
Region-Directed Enzyme Immobilization through Engineering Protein Surface with Histidine Clusters. ACS Appl. Mater. Interfaces
2024, 16, 833–846. [CrossRef]
83. Mateo, C.; Fernandez-Lorente, G.; Cortes, E.; Garcia, J.L.; Fernandez-Lafuente, R.; Guisan, J.M. One-step Purification, Covalent
Immobilization, and Additional Stabilization of poly-His-tagged Proteins Using Novel Heterofunctional Chelate-Epoxy Supports.
Biotechnol. Bioeng. 2001, 76, 269–276. [CrossRef] [PubMed]
84. Li, M.; Cheng, F.; Li, H.; Jin, W.; Chen, C.; He, W.; Cheng, G.; Wang, Q. Site-Specific and Covalent Immobilization of His-Tagged
Proteins via Surface Vinyl Sulfone–Imidazole Coupling. Langmuir 2019, 35, 16466–16475. [CrossRef]
85. Long, J.; Li, X.; Liu, X.; Jin, Z.; Xie, Z.; Xu, X.; Lu, C. Preparation of Streptavidin-Coated Magnetic Nanoparticles for Specific
Immobilization of Enzymes with High Activity and Enhanced Stability. Ind. Eng. Chem. Res. 2021, 60, 1542–1552. [CrossRef]
86. Liu, X.; Li, X.; Xie, Z.; Zhou, X.; Chen, L.; Qui, C.; Lu, C.; Jin, Z.; Long, J. Comparative Study on Different Immobilization Sites of
Immobilized β-Agarase Based on the Biotin/Streptavidin System. Int. J. Biol. Macromol. 2024, 261, 129807. [CrossRef] [PubMed]
87. Cronan, J.E. Biotination of Proteins in vivo. A Post-Translational Modification to Label, Purify, and Study Proteins. J. Biol. Chem.
1990, 265, 10327–10333. [CrossRef] [PubMed]
88. Cull, M.G.; Schatz, P.J. Biotinylation of Proteins in vivo and in vitro Using Small Peptide Tags. Methods Enzymol. 2000, 326,
430–440. [CrossRef]
89. Vishwanath, S.; Bhattacharyya, D.; Huang, W.; Bachas, L.G. Site-Directed and Random Enzyme Immobilization on Functionalized
Membranes: Kinetic Studies and Models. J. Membr. Sci. 1995, 108, 1–13. [CrossRef]
90. Alftrén, J.; Ottow, K.E.; Hobley, T.J. In vivo Biotinylation of Recombinant Beta-Glucosidase Enables Simultaneous Purification
and Immobilization on Streptavidin Coated Magnetic Particles. J. Mol. Catal. B Enzym. 2013, 94, 29–35. [CrossRef]
Molecules 2025, 30, 939 32 of 33

91. Benítez-Mateos, A.I.; Paradisi, F. Sustainable Flow-synthesis of (Bulky) Nucleoside Drugs by a Novel and Highly Stable Nucleoside
Phosphorylase Immobilized on Reusable Supports. ChemSusChem 2022, 15, e202102030. [CrossRef] [PubMed]
92. Hoelzel, C.A.; Zhang, X. Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies.
ChemBioChem 2020, 21, 1935–1946. [CrossRef]
93. Döbber, J.; Pohl, M. HaloTagTM : Evaluation of a Covalent One-Step Immobilization for Biocatalysis. J. Biotechnol. 2017, 241,
170–174. [CrossRef] [PubMed]
94. Döbber, J.; Gerlach, T.; Offermann, H.; Rother, D.; Pohl, M. Closing the Gap for Efficient Immobilization of Biocatalysts in
Continuous Processes: HaloTagTM Fusion Enzymes for a Continuous Enzymatic Cascade towards a Vicinal Chiral Diol. Green
Chem. 2018, 20, 544. [CrossRef]
95. Seide, S.; Arnold, L.; Wetzels, S.; Bregu, M.; Gätgens, J.; Pohl, M. From Enzyme to Preparative Cascade Reactions with Immobilized
Enzymes: Tuning Fe(II)/α-Ketoglutarate-Dependent Lysine Hydroxylases for Application in Biotransformations. Catalysts 2022,
12, 354. [CrossRef]
96. Zakeri, B.; Fierer, J.O.; Celik, E.; Chittock, E.C.; Schwarz-Linek, U.; Moy, V.T.; Howarth, M. Peptide Tag Forming a Rapid Covalent
Bond to a Protein, through Engineering a Bacterial Adhesin. Proc. Natl. Acad. Sci. USA 2012, 24, E690–E697. [CrossRef] [PubMed]
97. Keeble, A.H.; Banerjee, A.; Ferla, M.P.; Reddington, S.C.; Khairil Anuar, I.N.A.; Howarth, M. Evolving Accelerated Amidation by
SpyTag/SpyCatcher to Analyze Membrane Dynamics. Angew. Chem. Int. Ed. 2017, 56, 16521–16525. [CrossRef]
98. Veggiani, G.; Nakamura, T.; Brenner, M.D.; Gayet, R.V.; Yan, J.; Robinson, C.V.; Howarth, M. Programmable Polyproteams Built
Using Twin Peptide Superglues. Proc. Natl. Acad. Sci. USA 2016, 113, 1202–1207. [CrossRef] [PubMed]
99. Tan, L.L.; Hoon, S.S.; Wong, F.T. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE
2016, 11, e0165074. [CrossRef]
100. Khairil Anuar, I.N.A.; Banerjee, A.; Keeble, A.H.; Carella, A.; Nikov, G.I.; Howarth, M. Spy&Go Purification of SpyTag-Proteins
Using Pseudo-SpyCatcher to Access an Oligomerization Toolbox. Nat. Commun. 2019, 10, 1734. [CrossRef]
101. Lin, Z.; Lin, Q.; Li, J.; Pistolozzi, M.; Zhao, L.; Yang, X.; Ye, Y. Spy Chemistry-enabled Protein Directional Immobilization and
Protein Purification. Biotechnol. Bioeng. 2020, 117, 2923–2932. [CrossRef] [PubMed]
102. Gao, X.; Wei, C.; Qi, H.; Li, C.; Lu, F.; Qin, H.-M. Directional Immobilization of D-Allulose 3-Epimerase Using SpyTag/SpyCatcher
Strategy as a Robust Biocatalyst for Synthesizing D-Allulose. Food Chem. 2023, 402, 134199. [CrossRef] [PubMed]
103. Zhang, L.; Wang, W.; Yang, Y.; Liu, X.; Zhu, W.; Pi, L.; Liu, X.; Wang, S. Spontaneous and Site-Specific Immobilization of PNGase
F via Spy Chemistry. RSC Adv. 2023, 13, 28493. [CrossRef] [PubMed]
104. Parthasarathy, R.; Subramanian, S.; Boder, E.T. Sortase A as a Novel Molecular “Stapler” for Sequence-Specific Protein Conjugation.
Bioconjug. Chem. 2007, 18, 469–476. [CrossRef]
105. Hata, Y.; Matsumoto, T.; Tanaka, T.; Kondo, A. C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated
Technique. Macromol. Biosci. 2015, 15, 1375–1380. [CrossRef] [PubMed]
106. Qafari, S.M.; Ahmadian, G.; Mohammadi, M. One-Step Purification and Oriented Attachment of Protein A on Silica and Graphene
Oxide Nanoparticles Using Sortase-Mediated Immobilization. RSC Adv. 2017, 7, 56006. [CrossRef]
107. Moosavi, F.; Ahrari, F.; Ahmadian, G.; Mohammadi, M. Sortase-Mediated Immobilization of Candida antarctica Lipase B (CalB) on
Graphene Oxide; Comparison with Chemical Approach. Biotechnol. Rep. 2022, 34, e00733. [CrossRef]
108. Ito, T.; Sadamoto, R.; Naruchi, K.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.-I. Highly Oriented Recombinant
Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A.
Biochemistry 2010, 49, 2604–2614. [CrossRef]
109. Strop, P. Versatility of Microbial Transglutaminase. Bioconjug. Chem. 2014, 25, 855–862. [CrossRef] [PubMed]
110. Oteng-Pabi, S.K.; Clouthier, C.M.; Keillor, J.W. Design of a Glutamine Substrate Tag Enabling Protein Labelling Mediated by
Bacillus subtilis Transglutaminase. PLoS ONE 2018, 13, e0197956. [CrossRef]
111. Steffen, W.; Ko, F.C.; Patel, J.; Lyamichev, V.; Albert, T.J.; Benz, J.; Rudolph, M.G.; Bergmann, F.; Streidl, T.; Kratzsch, P.;
et al. Discovery of a Microbial Transglutaminase Enabling Highly Site-Specific Labeling of Proteins. J. Biol. Chem. 2017, 292,
15622–15635. [CrossRef]
112. Kamiya, N.; Doi, S.; Tanaka, Y.; Ichinose, H.; Goto, M. Functional Immobilization of Recombinant Alkaline Phosphatases Bearing
a Glutamyl Donor Substrate Peptide of Microbial Transglutaminase. J. Biosci. Bioeng. 2007, 104, 195–199. [CrossRef] [PubMed]
113. Moriyama, K.; Sung, K.; Goto, M.; Kamiya, N. Immobilization of Alkaline Phosphatase on Magnetic Particles by Site-Specific and
Covalent Cross-Linking Catalyzed by Microbial Transglutaminase. J. Biosci. Bioeng. 2011, 111, 650–653. [CrossRef] [PubMed]
114. Yu, C.-M.; Zhou, H.; Zhang, W.-F.; Yang, H.-M.; Tang, J.-B. Site-Specific, Covalent Immobilization of BirA by Microbial Transglu-
taminase: A Reusable Biocatalyst for in vitro Biotinylation. Anal. Biochem. 2016, 511, 10–12. [CrossRef] [PubMed]
115. Wang, J.-H.; Tang, M.-Z.; Yu, X.-T.; Xu, C.-M.; Yang, H.-M.; Tang, J.-B. Site-Specific, Covalent Immobilization of an Engineered
Enterokinase onto Magnetic Nanoparticles through Transglutaminase-Catalyzed Bioconjugation. Colloids Surf. B Biointerfaces
2019, 117, 506–511. [CrossRef]
Molecules 2025, 30, 939 33 of 33

116. Zhang, L.; Wang, W.; Yang, Y.; Zhu, W.; Li, P.; Wang, S.; Liu, X. Site-Specific, Covalent Immobilization of PNGase F on Magnetic
Particles Mediated by Microbial Transglutaminase. Anal. Chim. Acta 2023, 1250, 340972. [CrossRef]
117. Serra, I.; Ubiali, D.; Cecchini, D.A.; Calleri, E.; Albertini, A.M.; Terreni, M.; Temporini, C. Assessment of Immobilized PGA
Orientation via the LC-MS Analysis of Tryptic Digests of the Wild Type and Its 3K-PGA Mutant Assists in the Rational Design of
a High-Performance Biocatalyst. Anal. Bioanal. Chem. 2013, 405, 745–753. [CrossRef] [PubMed]
118. Pagolu, R.; Singh, R.; Shanmugam, R.; Kondaveeti, S.; Patel, S.K.S.; Kalia, V.C.; Lee, K.-K. Site-Directed Lysine Modification of
Xylanase for Oriented Immobilization onto Silicon Dioxide Nanoparticles. Bioresour. Technol. 2021, 331, 125063. [CrossRef]
119. Grazu, V.; Lopez-Gallego, F.; Montes, T.; Abian, O.; Gonzalez, R.; Hermoso, J.A.; Garcia, J.L.; Mateo, C.; Guisan, J.M. Promotion of
Multipoint Covalent Immobilization through Different Regions of Genetically Modified Penicillin G Acylase from E. coli. Process
Biochem. 2010, 45, 390–398. [CrossRef]
120. Hong, V.; Presolski, S.I.; Ma, C.; Finn, M.G. Analysis and Optimization of Copper-Catalyzed Azide–Alkyne Cycloaddition for
Bioconjugation. Angew. Chem. Int. Ed. 2009, 48, 9879–9883. [CrossRef] [PubMed]
121. Wu, J.C.Y.; Hutchings, C.H.; Lindsay, M.J.; Werner, C.J.; Bundy, B.C. Enhanced Enzyme Stability through Site-Directed Covalent
Immobilization. J. Biotechnol. 2015, 193, 83–90. [CrossRef]
122. Wang, A.; Du, F.; Pei, X.; Chen, C.; Wu, S.G.; Zheng, Y. Rational Immobilization of Lipase by Combining the Structure Analysis
and Unnatural Amino Acid Insertion. J. Mol. Catal. B Enzym. 2016, 132, 54–60. [CrossRef]
123. Guan, D.; Kurra, Y.; Liu, W.; Chen, Z. A Click Chemistry Approach to Site-Specific Immobilization of a Small Laccase Enables
Efficient Direct Electron Transfer in a Biocathode. Chem. Commun. 2015, 51, 2522. [CrossRef]
124. Bednar, R.M.; Golbek, T.W.; Kean, K.M.; Brown, W.J.; Jana, S.; Baio, J.E.; Karplus, P.A.; Mehl, R.A. Immobilization of Proteins with
Controlled Load and Orientation. ACS Appl. Mater. Interfaces 2019, 11, 36391–36398. [CrossRef] [PubMed]
125. Lampkowski, J.S.; Villa, J.K.; Young, T.S.; Young, D.D. Development and Optimization of Glaser–Hay Bioconjugations. Angew.
Chem. Int. Ed. 2015, 54, 9343–9346. [CrossRef]
126. Switzer, H.J.; Howard, C.A.; Halonski, J.F.; Peairs, E.M.; Smith, N.; Zamecnik, M.P.; Verma, S.; Young, D.D. Employing Non-
Canonical Amino Acids towards the Immobilization of a Hyperthermophilic Enzyme to Increase Protein Stability. RSC Adv. 2023,
13, 8496. [CrossRef] [PubMed]

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