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\DOI{10.5802/crchim.386}
\datereceived{2024-11-11}
\daterevised{2025-02-14}
\dateaccepted{2025-02-19}
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\dateposted{2025-04-07}
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\title{Biocatalysis in packed-bed reactors: immobilization as an
enabling technology} 

\alttitle{Biocatalyse dans les r\'eacteurs \`a lit fixe : l'immobilisation comme technologie cl\'e}

\author{\firstname{Cristina L\'{i}a} \lastname{Fern\'{a}ndez Regueiro}\CDRorcid{0000-0001-6709-2550}}
\address{inSEIT AG, Gesellschaftsstrasse 42, 3012 Bern, Switzerland}

\author{\firstname{David} \lastname{Roura Padrosa}\CDRorcid{0000-0001-6324-8722}\IsCorresp}
\addressSameAs{1}{inSEIT AG, Gesellschaftsstrasse 42, 3012 Bern, Switzerland}
\email[D. Roura Padrosa]{david.roura@inseit.ch}

\author{\firstname{Francesca}\nobreakauthor\lastname{Paradisi}\CDRorcid{0000-0003-1704-0642}\IsCorresp}
\address{Department of Chemistry, Biochemistry and Pharmaceutical
Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland}
\email[F. Paradisi]{francesca.paradisi@unibe.ch}

\keywords{\kwd{Biocatalysis}
\kwd{Enzyme immobilization}
\kwd{Flow biocatalysis}
\kwd{Packed-bed reactors}}

\altkeywords{\kwd{Biocatalyse}\kwd{Immobilisation enzymatique}\kwd{Biocatalyse en flux}\kwd{R\'eacteurs \`a lit fixe}} 

\begin{abstract}
Biocatalysis and flow chemistry are two complementary technologies that
can be used to produce chemicals in a more efficient, sustainable, and
safe manner. Enzyme immobilization is key to enabling their combination.
Various immobilization methods, including carrier-free and
carrier-based techniques, offer distinct advantages in terms of enzyme
activity, stability, and productivity. This review explores these
immobilization strategies and their integration in packed-bed reactors,
highlighting their industrial applications and potential challenges. 
\end{abstract}

\begin{altabstract}
La biocatalyse et la chimie en flux sont deux technologies
compl\'ementaires qui peuvent \^etre utilis\'ees pour produire des
compos\'es chimiques de mani\`ere plus efficace, plus durable et plus
s\^ure. Pour permettre leur combinaison, l'immobilisation des enzymes
est essentielle. Diverses m\'ethodes d'immobilisation, dont des
techniques sans et \`a base de support, offrent des avantages distincts
en termes d'activit\'e, de stabilit\'e et de productivit\'e de
l'enzyme. Cette revue explore ces strat\'egies d'immobilisation et leur
int\'egration dans les r\'eacteurs \`a lit fixe, en mettant en
\'evidence leurs applications industrielles et les d\'efis potentiels
qu'elles posent.
\end{altabstract}

\shortrunauthors

\maketitle
%{\vspace*{2pc}}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{mark-1.}

{Enzymes, nature's catalysts, have evolved to facilitate biochemical
reactions with remarkable specificity. Historically, they have been
harnessed in fermentation processes for food and beverage
production}~\cite{bib1,bib2,bib3,bib4}, {and early industrial
applications were predominantly focused on the use of naturally
occurring enzymes like xylose isomerase for high-fructose corn syrup
production}~\cite{bib5}, {nitrile hydratase for acrylamide
synthesis}~\cite{bib6,bib7}, {and penicillin amidase for semisynthetic
penicillins such as ampicillin}~\cite{bib8}. {Their high selectivity
avoided the need for complex protective group strategies, thereby
simplifying processes, reducing waste, and aligning with the increasing
demand for sustainable industrial practices.}


{Despite these examples, natural enzymes often exhibited limitations
for industrial-scale application. Their narrow substrate scope and
suboptimal \mbox{operational} stability in intensified conditions often
hindered broader industrial adoption}~\cite{bib2}. {Advancements in
directed evolution}~\cite{bib9,bib10} {and protein
engineering}~\cite{bib11} {have significantly mitigated these
limitations. There are examples of evolved enzymes with enhanced
solvent resistance}~\cite{bib12}, {improved stability under process
conditions, optimized pH and temperature ranges, and even more
importantly, where their reactivity is tailored to the substrate of
interest while maintaining excellent enantioselectivity}~\cite{bib13}.  

{In parallel to the growth of biocatalysis, flow chemistry has become
increasingly important over the past few decades. Unlike traditional
batch processes, flow chemistry allows for more precise control of the
reaction conditions (temperature, pressure, contact time between
substrate and catalyst), resulting in higher yields, safer operations,
and reduced waste}~\cite{bib14,bib15,bib16}. {Recently, the interest in
flow strategies has grown significantly in the fine chemical
industry}~\cite{bib17,bib18,bib19,bib20,bib21}. {Among the multiple
reactor types, packed-bed reactors (PBRs) allow the confinement of the
catalyst onto a column or similar reactor type. 
The confinement of the catalyst ensures a clean out-stream, 
facilitating downstream processing and product purification. 
Moreover, while scale-up is possible, PBRs also
allow for the scale-out strategy---the use of parallel small-scale
reactors to process larger volumes in shorter times. For this, PBRs
hold exciting potential in the synthesis optimization of fine
chemicals, while reducing costs and performing consistently, in a more
sustainable manner, making flow chemistry essential for modern,
sustainable chemical production.}

{Looking at the advantages of both technologies, it is only logical to
combine them; this is the objective of flow biocatalysis. However,
even engineered enzymes have a major downside for their integration
into continuous manufacturing: their reusability. Enzymes are
water-soluble and act in solution, and due to their complex structure,
their recycling and reuse for multiple batches is extremely 
difficult---if not impossible---without modification. To solve this problem, enzyme
immobilization is crucial to enabling enzymes in continuous reactors
(Figure~\ref{fig1}). Immobilization is a simple yet extremely powerful technique
that involves confining enzymes within or onto solid materials,
creating a heterogeneous biocatalyst that is no longer in solution.
Immobilized enzymes allow for their reuse, and enhance the
cost-efficiency of enzymatic processes}~\cite{bib3,bib22}. {Importantly,
immobilized enzymes often exhibit enhanced stability due to the reduced
flexibility upon immobilization and can retain activity over extended
periods of time, even under conditions that normally would inactivate
enzymes in solution}~\cite{bib3,bib23}. {Immobilized enzymes have been
used in the pharmaceutical, fine chemical, and food industries, and
yet only a few examples of their use in PBRs are
available.}

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig01}
{\vspace*{-3pt}}
\caption{\label{fig1}Main advantages of enzyme immobilization and continuous manufacturing.}
{\vspace*{-2pt}}
\end{figure*}

{Several reviews have addressed the evolution of flow biocatalysis
during the past 15 years}~\cite{bib24,bib25,bib26,bib27}. In this
concise review, we have focused on the applications of distinct enzyme
immobilization techniques in the form of PBRs. We discuss the different
immobilization methods and highlight their applications in industrially
relevant transformation in continuous flow, discussing their advantages and
the challenges encountered. 
By understanding these developments, we aim
to highlight the potential for future innovations that can further
integrate enzymatic processes into continuous manufacturing for
industrial chemistry.\looseness=-1



\section{Immobilization techniques and their application in 
continuous-flow setups}

{Since the discovery of enzyme immobilization by Nelson and
Griffith}~\cite{bib28}, {many different approaches have been developed
for the immobilization of enzymes. Finding a universal method has been
proven challenging due to the complex nature of the enzymes and the
multiple factors that play a role in the final activity and stability
of the final biocatalyst. Thus, in most cases, the immobilization of
choice is tailored to a specific enzyme for a desired
application.}\looseness=-1 

Immobilization methods are typically classified into reversible
techniques (such as adsorption, encapsulation, and reversible covalent
binding) and irreversible techniques (including cross-linking and
irreversible covalent binding). However, for the purpose of this short
overview, we classify immobilization methods based on the use (or
not) of a solid support to immobilize the enzyme: carrier-based and
carrier-free methods (Figure~\ref{fig2}).


\begin{figure*}
\includegraphics{fig02}
{\vspace*{-3pt}}
\caption{\label{fig2}Schematic representation of the different methods
of immobilization that have been used for continuous-flow
biocatalysis.}
{\vspace*{-2pt}}
\end{figure*}


\subsection{Carrier-free immobilized enzymes}

{Carrier-free immobilization of enzymes is carried out through their
cross-linking to form insoluble supramolecular structures.
Cross-linking involves the formation of covalent bonds between enzymes
using bifunctional or multifunctional reagents like glutaraldehyde.
These methods aim to maximize enzyme loading on the final biocatalyst
by eliminating the need for carriers and maximizing the activity per
unit of mass. However, they also present significant drawbacks that
limit their applicability, especially, their mechanical stability and
reusability}~\cite{bib29,bib30}.\looseness=-1 


{There are different approaches to the creation of cross-linked (CL)
biocatalysts. Enzymes can be cross-linked in different forms: as
soluble enzymes (CLEs), lyophilized (CLELs), or in the form of enzyme
aggregates (cross-linked enzyme aggregates [CLEAs]) and enzyme crystals (CLECs)}~\cite{bib30}. {Among
these options, the most common strategies are CLEAs and CLECs, but only
CLECs have been successfully incorporated into PBRs without further
modification}~\cite{bib31}. {The application of CLEAs in flow
biocatalysis has been challenging due to two main issues: the
scalability in their production and their mechanical
fragility}~\cite{bib31,bib32,bib33}. {Nonetheless, there are some
examples such as the ${\upgamma}$-lactamase CLEAs employed in a flow
microreactor built from a capillary column}~\cite{bib34,bib35}. 

\begin{figure*}
\includegraphics{fig03}
{\vspace*{-2pt}}
\caption{\label{fig3}Selected examples of the use of CLECs in PBR systems.}
\end{figure*}

{On the other hand, CLECs show higher mechanical resistance and have
been applied in different chemical reactor
configurations}~\cite{bib36,bib37}. {Although only a few examples are
available, their higher mechanical stability has allowed their
successful use in PBRs (Figure~\ref{fig3}). For example, glucoamylase
CLECs have been used for the continuous production of glucose from
starch and maltodextrin in PBRs}~\cite{bib38}. {In a more recent
example, the preparation of different ${\upbeta}$-substituted alcohols
using halohydrin dehalogenase HheG CLECs has been successfully scaled
up}~\cite{bib32}.  The production of HheG CLECs was performed using a
stirred crystallization approach, enabling upscaling beyond 50 mL. The
packing of these CLECs in a PBR facilitated the production of
2-azidocyclohexan-1-ol with an impressive space--time yield of
$28.7~\mathrm{kg}{\cdot}\mathrm{L}^{-1}{\cdot}\mathrm{day}^{-1}$,
demonstrating excellent operational stability and efficiency, which
\mbox{highlights} their potential for industrial applications in
continuous-flow  processes.

\subsection{Carrier-based methods}

Carrier-based methods involve the use of a matrix or solid support that
interacts with the enzymes and produces water-insoluble materials. The
range of materials that have been already used is broad and expanding,
including natural polymers, liposomes, petroleum-based polymers,
inorganic materials, and more recently, metal-organic frameworks.
The interaction of the enzyme with the carrier can be through
physical confinement (entrapment and encapsulation), non-covalent
interactions (adsorption, ionic interactions), or covalent bonding.
Each of the strategies has its benefits and downsides. When using
either entrapment or non-covalent interactions, the activity tends to be
unaffected, but the reusability and stability of the resulting
biocatalyst are generally low, except in specific enzyme types such as
lipases, making its incorporation into continuous manufacturing
processes challenging. On the other hand, with covalent methods, the
reduced flexibility of the enzyme affects its activity but offers
preparations with much higher stability and reusability. 

\subsubsection{Entrapment and encapsulation methods}

{Entrapment and encapsulation methods leverage a physical restriction
of the enzymes or cells within a confined space or network using a
carrier, in this case, a matrix. Several reviews focus on the
techniques and discuss the applicability and limitations of these
methods}~\cite{bib39,bib40}. {In general, entrapment provides a
protective environment for the enzyme, but the presence of a surrounding 
\mbox{extra}
layer causes diffusional limitations. Moreover, if the pore size
of the matrix is bigger than the enzyme size, potential leakage might
happen during the reaction.} 


{Probably the most common method of encapsulation of enzymes is
alginate hydrogels}~\cite{bib41,bib42}. {Since the 1990s, a lot of
research has focused on this method for its easy, cost-efficient, and
mild conditions for preparation. Carefully controlling the conditions
of polymerization, precursors, and modifiers allows for tailoring
material porosity, which is especially important. Nonetheless, alginate
encapsulation often suffers from low recyclability, a key aspect for
applying biocatalysis in PBRs. Two \mbox{different} approaches have been taken
to solve these problems: a post-entrapment treatment of the
alginate beads and the entrapment of supramolecular enzyme structures
(CLEAs, CLECs, and others).} 


\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig04}
{\vspace*{1pt}}
\caption{\label{fig4}Selected examples of the use of entrapment and
encapsulation methods in PBR systems.  If specified in the original
work, the contact time (${\upvarphi}$) or the amount of biocatalyst
used is indicated together with the space--time yield in g/L/h and
either stability or productivity.}
\end{figure*}

{One of the simplest post-modifications to alginate beads that has
shown advantages is their drying. 
The so-called dried alginate-entrapped enzymes have been used in 
continuous flow for the
production of fructo-oligosaccharides starting from sucrose (Figure~\ref{fig4}).
This strategy yielded a process with excellent productivity with a
cheap and cost-effective immobilization method while keeping 
enzyme leakage minimal by selecting the appropriate process conditions
(especially the control of water content in reaction to avoid
rehydration)}~\cite{bib43}. {More recently, coating strategies with
various polymers have also been tested to stabilize the alginate beads
further, increase their reusability, and prevent the leaching of the
enzyme. In some cases, the resulting preparations have been applied in
PBRs, mostly as proof-of-concept reactions to show their applicability
and advantages in terms of stability and
reusability}~\cite{bib44,bib45}.  {Although these strategies have been
successful, the addition of a second layer hampers the free diffusion
of the substrate and product from the bulk of the reaction to the
biocatalyst. On the other hand, there are only a few examples of the
use of supramolecular enzyme structures entrapped in alginate in
PBRs}~\cite{bib46,bib47}.

{Another interesting option for encapsulation is
Lentikats}$^{\text{\textregistered}}$. Reported 20 years ago,
Lentikats$^{\text{\textregistered}}$ {mediated encapsulation relies on
the formation of polyvinyl acetate hydrogels under mild conditions,
harnessing the excellent mechanical stability that this polymer offers.
Interestingly, the cross-linking of the enzyme is essential for
entrapment to be efficient in Lentikats}$^{\text{\textregistered}}$
as discussed even in the first reports~\cite{bib48}. {Several samples
have been incorporated into PBRs (Figure~\ref{fig4}). Guajardo} 
{{et~al}}.~\cite{bib49} {used a combination of CLEA and
Lentikats}$^{\text{\textregistered}}$ {entrapment to allow for the use
of a lipase (CALB) in deep eutectic solvents, which served both as the
reaction media and cosubstrate. First, by preparing CLEA--CALB followed
by encapsulation in Lentikats}$^{\text{\textregistered}}${, they created
a biocatalyst that could be incorporated into a PBR and exhibited
higher reusability (${&gt;}$65\%) after six cycles of 24~h at
60~{\textdegree}C when compared to CLEA--CALB alone.}\looseness=-1 


{More recently, with the rise of additive manufacturing (3D printing)
for biocatalysis}~\cite{bib50}, Rabe \mbox{et~al.} investigated the use of
entrapped enzymes in a 3D printed agarose matrix and their application
in PBRs~\cite{bib51}. {In their initial work, a keto-isovalerate
decarboxylase mutant with enhanced thermal stability was printed in
disks created with agarose. The resulting materials were stacked within
a column reactor and used in the continuous production of
isobutyraldehyde. The resulting entrapped enzyme retained 36\% of the
activity and could be used for over 5~h continuously with \mbox{minimal}
elution of the enzyme (0.7\%). The same technology was used by Croci}
{{et~al}}.~\cite{bib52}, but this time for the co-immobilization
of an amine dehydrogenase (LE-AmDH-v1) and a cofactor regeneration
partner (CbFDH). In their study, the authors compared different
immobilization techniques highlighting the necessity to tailor the
immobilization process to the enzymatic system. Entrapment within the
agarose matrices was, in their case, the best solution to avoid enzyme
inactivation upon \mbox{immobilization} and to maintain the stability
of the immobilized preparation. When used in flow, it achieved up to 
$7.4~\mathrm{g}{\cdot}\mathrm{L}^{-1}{\cdot}\mathrm{day}^{-1 }$ of
productivity  and a total turnover  of
3239~mol$_{\mathrm{product}}$/mol$_{\mathrm{enzyme}}$.\looseness=1

\subsubsection{Carrier-bound immobilized enzymes for flow applications}

Carrier-binding methods involve the interaction of enzymes with
water-insoluble materials, such as natural polymers and inorganic
materials. This binding can be through physical adsorption, 
or covalent bonding.

\paragraph*{Physical adsorption}


Physical adsorption is one of the simplest and the earliest methods
used for enzyme immobilization. It involves the attachment of enzymes
onto carriers through weak forces such as van der Waals forces, ionic
interactions, and/or hydrogen bonding. This method typically does not
alter the native structure of the enzyme, preserving most of its
catalytic activity~\cite{bib53}. {This method was first described in
1916 when Nelson and Griffin demonstrated that an invertase physically
adsorbed onto charcoal remained catalytically active}~\cite{bib28}.
{However, the weak nature of these interactions also means that it is
easy for the enzyme to leach, detaching from the support even under
gentle conditions, and, in most cases, does not significantly improve
enzyme stability}~\cite{bib54}. 


{Despite the limitations, enzyme adsorption has been widely used in
industrial processes and enabled continuous
production}~\cite{bib53,bib55}. {The most prominent example of this
immobilization strategy is lipases}~\cite{bib56}{, which have been
extensively reviewed earlier}~\cite{bib57,bib58}.  {These enzymes are
mostly used in biodiesel production and enantioselective
transesterification or hydrolysis of esters and amides.} 


{One example of adsorption immobilization in industrial flow
biocatalysis is  Lipozyme$^{\text{\textregistered}}$ TL IM from Novozymes, a lipase
from} {\textit{Thermomyces lanuginosus}} {(Figure~\ref{fig5}). The
enzyme is immobilized by ionic adsorption onto silicate supports.
Lipozyme$^{\text{\textregistered}}$ TL IM can be used in non-aqueous environments
as PBRs, as the hydrophilic carrier retains the necessary water to
maintain enzyme activity even in the presence of oils or organic
solvents. It has been successfully applied
in many industrial processes}~\cite{bib59}, {including 
inter-esterification of sesame oil and fully hydrogenated soybean oil
at 70~{\textdegree}C in continuous-flow PBRs,
demonstrating the practical applicability of adsorbed
biocatalysts}~\cite{bib60}. {Other lipases immobilized with similar
strategies have also been applied successfully. Of importance is
Novozym 435, which has been applied to a myriad of esterification and
\mbox{hydrolysis} reactions as a PBR}~\cite{bib61,bib62}. {Another industrial
use of adsorption immobilization is the production of allulose in
continuous flow using ketose-3-epimerase immobilized on an ion
exchange resin and integrated in a PBR}~\cite{bib63,bib64,bib65}. {This
method has led to significant cost reductions and even the
commercialization of DOLCIA PRIMA$^{\text{\textregistered}}$ Allulose, a
low-calorie sweetener.}

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Examples of the application of non-covalently
bound enzymes in a PBR system. If specified in the original work, the
contact time (${\upvarphi}$) or the amount of biocatalyst used is
indicated together with the space--time yield in g/L/h and either
stability or productivity.}
\end{figure*}

{Even multienzymatic systems have been immobilized using adsorption
techniques (Figure~\ref{fig5}). Co-immobilized lactate dehydrogenase
and formate dehydrogenase on a carbon support facilitated
ketone reduction with} {{in situ}} {cofactor recycling in a
continuous-flow PBR. 
This system achieved a space--time yield of
$11.2~\mathrm{g}{\cdot}\mathrm{L}^{-1}{\cdot}\mathrm{h}^{-1}$ in flow
compared to
0.23~$\mathrm{g}{\cdot}\mathrm{L}^{-1}{\cdot}\mathrm{h}^{-1}$ in batch
processes, demonstrating the higher productivity achievable using
low-flow reactor volumes (0.48~mL vs 24~mL in batch)}~\cite{bib66}.


{Transaminases have also been immobilized through affinity adsorption
using EziG{\texttrademark} materials. EziG{\texttrademark} is a hybrid controlled-pore
glass coated with a functionalized polymer, engineered for selective
metal-ion affinity immobilization of enzymes (Figure~\ref{fig5}). It is designed
for high loading of enzymes (up to 30\% w/w). In a study performed by
B\"{o}hmer} {{et~al}}{., two stereoselective complementary
${\upomega}$-transaminases were immobilized onto EziG{\texttrademark},
achieving 20\% w/w loading. These immobilized enzymes were then
utilized in a multigram-scale continuous-flow PBR for the production of
(S)-${\upalpha}$-MBA over 96 consecutive hours without any detectable
loss of enzymatic activity}~\cite{bib67,bib68}.{\looseness=-1}

\paragraph*{Covalent binding}

{Covalent immobilization involves the formation of stable covalent
bonds between enzyme functional groups---such as amino, carboxyl, and
hydroxyl groups---and those on a suitable carrier (Figure~\ref{fig6}).
This method has become important due to the strong linkages it forms
through multipoint attachment, which significantly reduces enzyme
leaching and enhances stability compared to non-covalent
adsorption-based methods}~\cite{bib69}. {The use of covalent binding in
enzyme immobilization has been reported in numerous
reviews}~\cite{bib3,bib58,bib70,bib71}.

\begin{figure*}
\includegraphics{fig06}
{\vspace*{-1.5pt}}
\caption{\label{fig6}Selected examples of covalently bound enzymes
applied in PBR. The contact time (${\upvarphi}$) is indicated together
with the space--time yield in g/L/h and either stability or
productivity.}
{\vspace*{-3.8pt}}
\end{figure*}

{An exemplary case of covalent immobilization in flow biocatalysis is
the enzymatic synthesis of L-pipecolic acid using covalently
immobilized lysine cyclodeaminase}~\cite{bib72}. {This study compared
the \mbox{reaction} between a SpinChem$^{\text{\textregistered}}$ reactor and a
PBR. Batch reactions with the enzyme in solution
resulted in low conversion yields and significant enzyme deactivation
over time. Conversely, when employing continuous-flow reactors, full
conversion was achieved within 90 min while maintaining high
operational stability. Additionally, the study compared different
immobilization methods, including physical adsorption and covalent
binding, revealing that covalent immobilization provided better
reusability by preventing enzyme leaching.}


{Another example is the biocatalytic synthesis of}
{${(R)}$}{-sitagliptin in a recirculating PBR. In
this case, a transaminase was co-immobilized with PLP inspired by the
creation of a self-sufficient biocatalyst by Ben\'{i}tez-Mateos}
{{et~al}}.~\cite{bib73}. {This biocatalyst maintained yields and
enantiomeric excess values above 90\% and 99\%, respectively, over 700 batch
reactions without the use of additional PLP. An overall space--time
yield of 40.0~g/L/h was obtained}~\cite{bib74}. 

{Additionally, the synthesis of} {(\textit{S})-}{nornicotine was
enhanced through the covalent co-immobilization of IRED and
NADP-dependent GDH on an epoxy resin within a PBR. This
system maintained substrate conversion rates above 99\% after 114~h of
uninterrupted operation and attained a space--time yield of 211.47~g/L/h,
which is 289.7 times higher than that of batch processes}~\cite{bib75}.


\section{Current trends and outlook}

The integration of enzymes into PBRs is highly
dependent on developing novel materials and immobilization techniques.
Due to the complexity of these immobilization methods as discussed in
this review, it is challenging to determine in advance which
immobilized biocatalyst will perform best in flow systems.

Consequently, immobilization strategies are approached on a
case-by-case basis. Ideally, the search should focus on strategies that
can (1) minimize enzyme desorption and deactivation, (2) enhance
substrate and product diffusion to maximize conversion, and (3)
simplify downstream processing. However, with current techniques, there
is always a balance between biocatalyst activity and stability. It is
true though that stability is often favored when applying enzymes in
PBRs. Although immobilized enzyme activity might be lower than
free enzyme activity, the high local concentration of enzymes within the column
allows for elevated substrate-to-catalyst ratios, resulting in higher
conversion efficiency. Moreover, the precise control of reaction
conditions within PBRs enables further process optimization.
Adjustments such as increasing temperature, substrate loading, and
implementing recirculation have been successfully employed to mitigate
the lower activities observed in immobilized enzymes.


{Innovation from material and chemical sides is still greatly
needed in enzyme immobilization. Although immobilization has a long
history, it remains predominantly based on interactions with charged
amino acids, particularly the most reactive ones---almost exclusively
cysteine and lysine or activated aspartic and glutamic acids.
Therefore, developing new chemistry that can exploit other chemical
handles is essential to ensure the process flexibility required for
tailored immobilization strategies. Additionally, a deeper
understanding of the interactions between enzymes and supports is
important for optimizing these new immobilization techniques. Without
this understanding, optimizing new immobilization strategies can be
significantly hindered. Several key parameters of the support matrix
are vital for maximizing the catalytic properties of immobilized
enzymes. Consequently, investigations into novel formulations of
bio-based polymers (such as lignocellulosic materials and innovative
plastics), covalent- and metal-organic frameworks, and
even solid materials previously regarded as waste are essential to
enhance immobilization strategies}~\cite{bib76,bib77,bib78}. 


{Moreover, synergistically combining multiple immobilization techniques
has shown promising results. Early examples included the combination of
cross-linking and encapsulation. More recently, the integration of
CLEAs with carrier-based
immobilization has been employed to develop CLEANs (cross-linked
enzyme-adhered nanoparticles), which have been applied to the synthesis
of fragrances}~\cite{bib79}. 


{Adopting advanced screening technologies is crucial to identifying
the most effective immobilization techniques. For example,
L\'{o}pez-Gallego and coworkers developed microtiter plate 
immobilization screening for high-throughput prototyping. This protocol
tests 17 different carriers functionalized with various reactive
groups in a 96-well plate \mbox{format,} simultaneously screening up to 21
immobilization protocols for up to 18 enzymes. By evaluating both
activity and stability, this method enables the rapid identification of
optimal immobilization chemistry, significantly accelerating the
development of efficient multienzyme systems}~\cite{bib80}.
{Integrating computational tools like CapiPy, a Python-based GUI
application, further enhances this workflow by rationalizing screening
results and facilitating informed decision-making for immobilization
strategies}~\cite{bib81}.

\section*{Declaration of interests}

{Cristina L\'{i}a Fern\'{a}ndez Regueiro and David Roura Padrosa are
affiliated with inSEIT AG, and Francesca Paradisi is one of its founding
members and its key scientific advisor.}

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