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\DOI{10.5802/crchim.397}
\datereceived{2024-11-22}
\daterevised{2025-03-12}
\dateaccepted{2025-03-26}
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\dateposted{2025-06-04}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\title{Innovative carrier materials for advancing enzyme immobilization
in industrial biocatalysis}

\alttitle{Mat\'{e}riaux supports innovants pour la promotion de
l'immobilisation des enzymes dans la biocatalyse industrielle}

\author{\firstname{Quentin} \lastname{Hanniet}\CDRorcid{0000-0002-6066-9297}}
\address{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}
\address{CarboZym SAS, Cap Alpha, 3 avenue de l'Europe - 34830 Clapiers, France}

\author{\firstname{Coline} \lastname{Mateos}}
\addressSameAs{1}{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}

\author{\firstname{Ludivine} \lastname{Onillon}}
\addressSameAs{1}{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}

\author{\firstname{Awilda} \lastname{Maccow}}
\addressSameAs{1}{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}

\author{\firstname{Thierry}\nobreakauthor\lastname{Gefflaut}\CDRorcid{0000-0002-4411-8233}}
\address{Universit\'{e} Clermont Auvergne, Clermont-Auvergne INP, CNRS,
Institut de Chimie de Clermont-Ferrand, 63000 Clermont-Ferrand, France}

\author{\firstname{M\'{e}lanie} \lastname{Hall}}
\address{Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria}

\author{\firstname{Tamara} \lastname{Reiter}\CDRorcid{0000-0002-8497-1693}}
\addressSameAs{4}{Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria}

\author{\firstname{M\'{e}lanie} \lastname{Bordeaux}\CDRorcid{0000-0002-5444-2950}}
\address{Fundacion Nicafrance, Finca La Cumplida, Matagalpa, Nicaragua}

\author{\firstname{Nicolas}\nobreakauthor\lastname{Brun}\CDRorcid{0000-0002-1013-311X}}
\addressSameAs{1}{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}
\addressSameAs{2}{CarboZym SAS, Cap Alpha, 3 avenue de l'Europe - 34830 Clapiers, France}

\author{\firstname{Jullien} \lastname{Drone}\IsCorresp}
\addressSameAs{1}{ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France}
\addressSameAs{2}{CarboZym SAS, Cap Alpha, 3 avenue de l'Europe - 34830 Clapiers, France}
\email[J. Drone]{jullien.drone@enscm.fr}

\shortrunauthors

\keywords{\kwd{Enzyme immobilization}
\kwd{Lipase}
\kwd{Transaminase}
\kwd{Old yellow enzyme}
\kwd{Hydrothermal carbonization}
\kwd{Biobased carrier}
\kwd{Industrial biocatalysis}}

\altkeywords{\kwd{Immobilisation enzymatique}
\kwd{Lipase}
\kwd{Transaminase}
\kwd{Old yellow enzyme}
\kwd{Carbonisation hydrothermale}
\kwd{Support biosourc\'{e}}
\kwd{Biocatalyse industrielle}}

\thanks{Univ. Montpellier (LabMUSE Chimie 2021), R\'{e}gion Occitanie
(Pre-maturation 2021), SATT AxLR (Pre-maturation 2021, Maturation
2023)}

\begin{abstract}
Industrial biotechnology stands at a turning point. While enzymes have
long promised green, efficient catalysis, their widespread adoption has
been held back by practical limitations. In this work, we introduce an
innovative family of sustainable carrier materials that redefine enzyme
immobilization. These materials are synthesized via a controlled
solvothermal process, using renewable sources of sugars and polyphenols
to create an interconnected porous structure with tunable surface
chemistry. This design enables strong enzyme binding through mechanisms
such as metal complexation, hydrophobic interactions, and hydrogen
bonding, offering high loading capacities and exceptional operational
stability. Developed with sustainability at their core, these materials
set new standards for both performance and environmental
responsibility, bridging the gap between laboratory research and
industrial applications in biocatalysis.
\end{abstract}

\begin{altabstract}
La biotechnologie industrielle se trouve \`{a} un tournant. Alors que
les enzymes promettent depuis longtemps une catalyse verte et efficace,
leur adoption g\'{e}n\'{e}ralis\'{e}e a \'{e}t\'{e} frein\'{e}e par des
limitations pratiques. Dans ce travail, nous pr\'{e}sentons une famille
innovante de mat\'{e}riaux support durables qui red\'{e}finissent
l'immobilisation des enzymes. Ces mat\'{e}riaux sont
synth\'{e}tis\'{e}s par synth\`{e}se solvothermale contr\^{o}l\'{e}e et
bas\'{e}e sur une mati\`{e}re premi\`{e}re biosourc\'{e}e tels que des
sucres et des polyph\'{e}nols pour g\'{e}n\'{e}rer une structure
poreuse interconnect\'{e}e avec une chimie de surface \`{a} fa\c{c}on.
Ce mat\'{e}riau permet une interaction forte avec les enzymes par des
m\'{e}canismes tels que la complexation des m\'{e}taux, les
interactions hydrophobes et les liaisons hydrog\`{e}ne, offrant des
capacit\'{e}s de charge \'{e}lev\'{e}es et une stabilit\'{e}
op\'{e}rationnelle exceptionnelle. D\'{e}velopp\'{e}s dans une optique
de durabilit\'{e}, ces mat\'{e}riaux \'{e}tablissent de nouvelles
normes de performance et de responsabilit\'{e} environnementale,
comblant ainsi le foss\'{e} entre la recherche en laboratoire et les
applications industrielles en biocatalyse.
\end{altabstract}

\maketitle

\vspace*{4pt}

\twocolumngrid

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\section{Introduction}\label{sec1}

Enzyme immobilization has emerged as a powerful technique to enhance
enzyme stability, reusability, and efficiency~\cite{1}. It represents a
crucial technological advancement in industrial biotechnology, bridging
the gap between the remarkable catalytic properties of enzymes and the
practical demands of industrial processes. This strategy, which has
evolved significantly since its first documented use in 1916~\cite{2},
has become instrumental in various industrial \mbox{sectors} and continues to
shape the future of sustainable chemical manufacturing.

The industrial application of enzymes spans numerous sectors,
demonstrating their versatility and importance in modern manufacturing
processes. Key industries utilizing enzymatic processes include food
processing~\cite{3}, textile manufacturing~\cite{4}, biodiesel
production~\cite{5}, biosensors~\cite{6}, paper \mbox{making}~\cite{7},
cosmetics~\cite{3}, therapeutic molecules~\cite{8} and, but not limited
to, chemical synthesis~\cite{9}.

To understand the scale of enzyme utilization in industry, consider
that glucose production alone involves the conversion of 10$^{9}$ tons
of corn starch annually using soluble amylases. Similarly, the glucose
isomerase process handles the conversion of 10$^{7}$ tons of glucose
per year to make high-fructose corn syrup, highlighting the massive
scale of these operations~\cite{1}. From the economical standpoint, the
global enzyme market was valued at USD 14.0 billion in 2024 and is
projected to reach USD 20.4 billion by 2029, recording a Compound
Annual Growth Rate (CAGR) of 7.8\%~\cite{10}.

However, their successful implementation is often hindered by several
inherent limitations. One major challenge is their operational
instability. Enzymes are susceptible to denaturation under the harsh
conditions prevalent in many industrial processes, such as extreme
temperatures and pH fluctuations. Their natural structures may not
withstand prolonged operational periods, leading to a decline in
catalytic activity. Furthermore, the recovery of free enzymes, which
act as homogeneous catalysts, presents a significant hurdle. Being
water-soluble, they cannot be easily separated from the reaction
mixture. Traditional recovery methods, like membrane processes, are
often costly and inefficient, making continuous processing difficult.
Another challenge arises from the tendency of enzymes to aggregate,
particularly in hydrophobic environments or near their isoelectric
point. This aggregation limits diffusion and reduces accessibility to
active sites, ultimately decreasing overall activity~\cite{11}. 

Enzyme immobilization technology presents a compelling solution to the
challenges associated with the use of free enzymes in industrial
processes~\cite{12}. By anchoring enzymes onto a solid support,
immobilization confers several key advantages. \looseness=1

Firstly, it transforms enzymes from homogeneous to heterogeneous
catalysts, enabling straightforward recovery through simple filtration
or centrifugation techniques. This eliminates the need for costly and
inefficient downstream processing methods, significantly reducing
overall process costs and improving efficiency.

Furthermore, immobilization significantly enhances enzyme
stability~\cite{13}. The immobilized enzymes exhibit greater resistance
to harsh \mbox{environmental} conditions, such as temperature fluctuations and
pH changes. This is translated to an extended operational lifetime and
a broader range of applicable reaction conditions. 

Moreover, immobilized enzymes are ideally suited for continuous flow
operations, allowing for seamless integration into packed or fluidized
bed reactors. This improved process control enables more precise
regulation of reaction parameters and enhances overall system
efficiency~\cite{14,15}. The choice of support material plays a crucial
role in the immobilization process, affecting the properties of the
resulting biocatalytic system~\cite{16}. A wide range of materials,
including inorganic, organic, hybrid, and composite supports, have been
explored for enzyme immobilization~\cite{16,17}. Additionally,
agroindustrial wastes have shown potential as cost effective and
environmentally friendly carriers 
for enzyme immobilization, coconut fiber being a
preferred\break option~\cite{18,19}.

While enzyme immobilization offers numerous benefits, the field still
faces certain challenges that limit its widespread adoption since no
single method is universally applicable. One key hurdle lies in the
complexity and time-consuming nature of immobilization
protocols~\cite{20}. Current methods often require lengthy procedures,
sometimes extending up to 48 h, involving the use of hazardous
chemicals such as glutaraldehyde and can result in variable protein
loading capacities, hindering efficient enzyme utilization. Moreover,
the recycling potential of immobilized enzymes may turn out to be
limited, requiring frequent replacement and contributing to increased
the operational costs.

Another challenge stems from the intrinsic limitations of support
materials. Many carriers currently used are expensive and/or rely on
oil-based materials, raising concerns about sustainability~\cite{21}.
\looseness=1

Furthermore, the performance of immobilized enzymes is often dependent
on the specific carrier used, leading to inconsistent results. Enzyme
activity retention can vary significantly, and protein loading capacity
remains limited, typically reaching around 5\% or 50 mg of enzyme per
gram of carrier~\cite{22}.\looseness=1

These limitations highlight the need for continued research and
development to optimize immobilization techniques and develop more
efficient, versatile and sustainable solutions.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Description of XPh and XCat CarboZym support
synthesis and chemical structure.}
\end{figure*}

\section{The CarboZym technology: a game changer}\label{sec2}

At CarboZym SAS\footnote{CarboZym SAS, a CNRS spin-off established in
November 2024, has set itself the mission to transform chemical
manufacturing through the power of enzymes.}, a multidisciplinary team
of researchers in chemistry, bioprocess engineering, and biocatalysis
collaborates to make enzyme-powered biomanufacturing both accessible
and cost-effective and to pave the way for a~more sustainable future
by streamlining enzyme integration in processes.


\subsection{Underlying principle}\label{sec21}

The CarboZym technology, which we will call \mbox{``CarboZym'',} marks a
significant advancement in biocatalysis by patenting a biosourced,
universally adaptable porous support for enzyme
immobilization~\cite{23,24}. This innovative support, derived from
renewable precursors such as sugars (xylose, glucose, fructose) and
phenols (phloroglucinol, catechin), leverages polycondensation
mechanisms and intermolecular
reactions~\cite{25}. Unique characteristics
of this material include an interconnected porous structure and
adjustable surface chemistry~\cite{26}, enabling various types of
interactions with enzymes. Relying on green chemistry principles and
biodegradable raw materials, this technology meets sustainability
requirements while minimizing carbon footprint, notably by revalorizing
bio-waste rich in phenolic compounds (such as condensed tannins).

\subsection{Material synthesis and properties}\label{sec22}

The preparation of the CarboZym support material involves a controlled
synthesis process in an autoclave under solvothermal, subcritical
conditions (Figure~\ref{fig1}). The process begins with dissolving the
precursors, namely a sugar and a phenol, in a water--ethanol mixture.
Under solvothermal conditions, the sugars undergo dehydration: xylose
is converted to furfural, while glucose and fructose generate
5-(hydroxymethyl)furfural (HMF). These heterocyclic aldehydes then
quickly react with phenols such as phloroglucinol or catechin (at
positions 2, 4, 6 of the aromatic ring) via intermolecular dehydration
and electrophilic aromatic substitution mechanisms~\cite{25}.

Initially, polymerization occurs between active sites of the phenols
and the dehydrated sugars, forming oligomers that gradually assemble
into three-dimensional networks through successive steps of nucleation,
particle growth, and agglomeration. Within the autoclave, these steps
promote the formation of a gel with interconnected pores (ranging from
microporosity (${&lt;}$2~nm) to macroporosity (a few micrometers)) filled
with liquid. This structure mimics sol--gel processes but is influenced
by the composition of the precursors and the applied solvothermal
conditions.\looseness=-1

The precursor solution solidifies during the reaction in the autoclave,
enabling the material to be shaped into various forms by placing a mold
in the autoclave (see Figure S1.a for a macroscopic view of the shaped
material). Alternatively, the material can be reduced to a powder after
post-drying milling for applications in fluidized systems.

Following gel formation, two crucial steps are required to replace the
interstitial liquid with air or another gas: solvent exchange through
intensive washing, and drying~\cite{27}. The choice of solvent and
drying method (supercritical drying, freeze drying or air drying) can
significantly affect the final material properties: 
\begin{enumerate}[(i)]
\item Supercritical drying is the method of choice as it yields
aerogels with full preservation of the original pore texture of the wet
gel. It is usually performed with carbon dioxide that presents moderate
critical point temperature and pressure (31.1~\textdegree C at
1072~psi). When performed correctly, supercritical drying can largely
preserve the material's textural properties. \looseness=-1

\item Freeze-drying produces cryogels and involves solvent exchange
with water or water--alcohol mixtures, freezing and ice sublimation at
low pressure (to the range of a few millibars). However, ice crystal
growth during the freezing stage can induce large distortion of the
porous structure and a drastic loss of porosity~\cite{25}. 

\item  Convective or evaporative air-drying is a much more delicate
method to use, since drying occurs by direct solvent evaporation and
the gel is therefore subject to high capillary forces. It yields
xerogels that often display slightly different yet functional
characteristics~\cite{27}. 
\end{enumerate}

This step allows the creation of materials with BET
(Brunauer--Emmett--Teller theory)-equivalent specific surface areas
ranging from 200 to 900~m$^{2}$/g and pore volumes between 0.2 and 
2~cm$^{3}$/g, with adjustable pore sizes between 20 and 70 nm,
depending on the synthesis conditions (Figure~S1.b shows adsorption and
desorption isotherms of XCat and XPh materials, and Figure~S1.c illustrates
their BJH (Barrett--Joyner--Halenda Model) pore distribution).
\vspace*{-5pt}

\subsection{Technology description}\label{sec23}

The flexibility of CarboZym material synthesis offers optimization
possibilities tailored to industrial needs. For instance, by selecting
catechin as the phenol and xylose as the sugar, the final material
(designated XCat) retains unaltered catechol functions, \unskip\break \mbox{allowing}
chelation with metal ions (see chemical structure in
Figure~\ref{fig1}). This option enables binding of His-tagged proteins
to the material through metal complex formation~\cite{28,29}. In the
presence of an ion such as nickel, cobalt, or iron, these catechol
groups form stable bonds with His-tagged enzymes, ensuring robust
fixation and precise enzyme orientation on the support
(Figure~\ref{fig2}b). This binding also facilitates enzyme recycling by
simply washing out the bound enzyme under mild conditions that disrupt
the His--Nickel interaction, by using imidazole, which is essential for
continuous-flow or industrial processes where the catalytic lifespan of
the enzyme is crucial.

\begin{figure*}
\includegraphics{fig02}
\vspace*{-6pt}
\caption{\label{fig2}(a) Protein confinement in the CarboZym support;
(b) Protein bonding through formation of a stable
XCat--Fe$^{3+}$--histidine complex; (c) Weak bonding of protein
through electrostatic interaction, Van der Waals forces,
$\uppi$--${\uppi}$ stacking and/or hydrophobic interaction.}
\vspace*{-6pt}
\end{figure*}

The CarboZym material also exhibits compatibility with non-His-tag
enzymes, which interact through weak bonds such as hydrophobic
interactions or hydrogen bons, enabled by functional groups present on
the porous surface. Thus, an XPh-type material (derived from xylose and
phloroglucinol, Figure~\ref{fig1}) would be suitable for non-His-tagged
proteins as it is more likely to interact with a protein via
hydrophobic interactions given its less oxygen-rich surface chemistry
compared to XCat. The material's adaptability in terms of surface and
chemistry makes it compatible with a wide range of enzymes, rendering
this technology potentially universal.

The enzymatic immobilization protocol is simplified compared to
conventional methods (such as cross-linking, entrapment, covalent
attachment) (Figure~\ref{fig2}a), where the enzyme self-confines
directly within the material meso- and macroporosity via physisorption
and/or chemisorption. At the molecular scale (Figure~\ref{fig2}b), the
immobilization of His-tagged enzymes appears to occur through metal
complexation with catechol groups in XCat gels, while non-His-tag
enzymes bind to the material through electrostatic interactions (via
lysine, histidine, or arginine residues), van der Waals forces,
${\uppi}$--${\uppi}$ stacking and/or hydrophobic interactions (e.g.,
between proline residue and polyphenol ring)~\cite{30,31,32}.

The sugars and phenols used in the synthesis of CarboZym supports
enhance its biosourced aspect. Catechin, for example, is a monomer of
condensed tannins, demonstrating the feasibility of using bio-waste
containing these compounds to design the gel. Revalorizing this waste
(such as wood or seed extracts) in the material's production not only
reduces environmental impact but also allows for sustainable and
economical precursor sourcing~\cite{33,34}.

\subsection{Green chemistry and industrial relevance}\label{sec24}

From an industrial standpoint, enzyme immobilization on CarboZym
carrier material offers several major advantages over the use of free
enzyme use. First, with the ability to immobilize up to 50~wt\% of
enzyme on the CarboZym material enables reusability, significantly
lowering the cost per usage cycle. Moreover, this technology allows
enzyme utilization in continuous mode, meeting the growing industrial
demand for flow optimization. However, technical challenges remain in
enzyme immobilization, such as activity loss due to diffusion
limitations or demanding attachment conditions.

CarboZym addresses limitations of traditional immobilization
technologies. The binding process is fast and spontaneous, without
requiring costly activation steps. The material's interconnected pores
enhance mass transfer, minimizing activity losses from diffusion
constraints. Additionally, enzyme reuse is assured, making this
approach compatible with moderately active enzymes, thereby avoiding
costly enzyme engineering optimizations. Finally, the biosourced nature
and green chemistry principles underlying this technology strengthen
its positioning in sustainable industrial applications.
\vspace*{-5pt}

\begin{figure*}
\includegraphics{fig03}
\vspace*{-5pt}
\caption{\label{fig3}Results for immobilized CALB (a) Batch mode:
conversion ratio and cumulative leaching for XPh and XCat support
([{\textit{buffer}}: 20~mM Tris-HCl ${+}$ 1~wt\% genapol; pH 7]
[{\textit{immobilization conditions}}: 10~mg XCat or XPh 
support---10\% (w/w) protein loading---800~rpm; 4~\textdegree C];
[{\textit{reaction conditions}}: 15~mM pNPB---25~\textdegree
C]);  (b) Continuous mode: conversion ratio and STY
([{\textit{buffer}}: same as batch mode]
[{\textit{immobilization conditions}}:  65 mg support---10\%
(w/w) protein loading---0.1~ml/min; 4~\textdegree C]; 
[{\textit{reaction conditions}}: same as batch mode; circulation
mode---5~mM p-NPB---13.3~ml at 0.5~ml/min---5~cycles]).}
\vspace*{-5pt}
\end{figure*}

\section{Illustrative case studies}\label{sec3}
\vspace*{-5pt}

\subsection{Tailoring \textit{Candida antarctica} lipase B (CALB)
performance through surface chemistry}\label{sec31}

Lipases are enzymes that catalyze the hydrolysis of ester bonds in
lipid molecules~\cite{35}. Their natural function is the breakdown of
fats, but they are also widely used in synthetic processes such as
esterification and transesterification, especially in non-aqueous
environments~\cite{36,37}. These enzymes are favored in biocatalysis
because of their broad substrate specificity, regioselectivity, and
stereoselectivity, making them valuable tools in the pharmaceutical,
food, and chemical industries. Lipases work at the interface between
aqueous and organic phases, and their activity is often enhanced by
this interfacial activation. One of the most prominent lipases in
industrial applications is \textit{Candida antarctica} lipase B (CALB).
This enzyme stands out due to its high stereoselectivity and substrate
flexibility, making it a key player in asymmetric synthesis,
particularly for secondary alcohols. CALB's robust catalytic
performance under various conditions, including in organic solvents, at
wide temperature and pH range, contributes to its broad use in
reactions such as polymerization, hydrolysis, and esterification.


Despite its numerous benefits, CALB's cost can be a significantly
limiting factor for large-scale industrial applications. To address
this, immobilization of CALB is often employed to enable enzyme reuse
and thus lower the overall production cost. However, the commercial
version of CALB, such as Novozyme 435, is already immobilized on a
resin. Despite its widespread use, one of the challenges associated
with this preparation is leaching---the gradual loss of enzyme from the
support during reactions. This leaching can diminish the enzyme's
activity over time, reducing the expected cost savings from
immobilization~\cite{38}. 

\begin{figure*}
\includegraphics{fig04}
\vspace*{-5pt}
\caption{\label{fig4}{Results for immobilized \textit{Gs}OYE}.
(a) Batch mode: conversion ratio and cumulative leaching
([{\textit{buffer}}: 200 mM Tris-HCl + 50 mM NaCl ${+}$ 2~vol\%
DMSO; pH 7.5] [{\textit{immobilization conditions}}: 25 mg
Fe-XCat support---10\% (w/w) protein loading---800 rpm; 4~\textdegree
C]; [{\textit{reaction conditions}}: 50 mM cyclohexenone---75
mM NADH---30~\textdegree C]); (b) Continuous mode: conversion ratio
and STY ([{\textit{buffer}}: same asbatch mode]
[{\textit{immobilization conditions}}: 50 mg Fe-XCat support---10\% 
(w/w) protein loading---0.1 ml/min; 4~\textdegree C];
[{\textit{reaction conditions}}: same as batch mode;
recirculation mode---120 ml at 1 ml/min---5~cycles]).}
\vspace*{-5pt}
\end{figure*}

The immobilization of CALB was carried out on two versions of CarboZym
support to form  biocatalysts $\mathbi{i}$-CALB-XCat and
$\mathbi{i}$-CALB-XPh. Such biocatalysts were implemented in the
hydrolysis of p-nitrophenyl butyrate (pNPB) as a model reaction (see
Figure~\ref{fig3} caption and supporting information for more detailed
protocol). Conversion ratio and cumulative leaching on 5 cycles
associated are displayed on Figure~\ref{fig3} for both biocatalysts. In
both cases, the enzyme is still active after 5 cycles but their
\mbox{remaining} activity is different, averaging 20 and 44\% for
$\mathbi{i}$-CALB-XCat and $\mathbi{i}$-CALB-XPh, respectively. One
explanation probably is linked to the fact that the cumulative leaching
over 5 cycles is almost 2 times greater for XCat than for XPh support.
According to these observations, XPh support is a more suitable
material for CALB immobilization as it is more difficult to remove the
enzyme from the material probably due to a stronger interaction. Our
hypothesis here is that the CALB interacts with the carbon-based
support through hydrophobic interactions which are stronger with XPh
than XCat.

The $\mathbi{i}$-CALB-XPh biocatalyst was then implemented in a
fluidized bed continuous flow system (see Supplementary Information) to prove the
implementation of CarboZym technology in such industry-approved
reactors. In continuous mode, the substrate solution was flowed
(without re-circulation) through the thermostatted column at 0.5 ml/min
with negligible protein loss and achieving space--time yields (STY)
close to 7~g${\cdot}$L$^{-1}{\cdot}\mathrm{h}^{-1}$ (for the p-NPB
hydrolysis). These results demonstrate significant progress toward
achieving the industry-standard metrics required to ensure process
viability~\cite{39}. 

\subsection{\mbox{\textit{Gs}OYE-catalyzed~reduction~of}\unskip\break 2-cyclohexenone to
cyclohexanone}\label{sec32}

Ene-reductases (EREDs) are a class of oxidoreductase enzymes that
catalyze the asymmetric reduction of activated alkenes to saturated
products, \mbox{typically} using nicotinamide cofactors such as NAD(P)H as
electron donors. Among the ene-reductases, the old yellow enzyme (OYE)
family is one of the most well-studied and widely applied groups. OYE
enzymes are flavin mononucleotide (FMN)-dependent oxidoreductases known
for their ability to catalyze the reduction of a wide variety of
${\upalpha}$,${\upbeta}$-unsaturated carbonyl compounds, including ketones,
aldehydes, and nitroalkenes~\cite{40,41}. OYE enzymes exhibit broad
substrate specificity and high stereoselectivity can be obtained,
making them valuable for applications in asymmetric synthesis. One
notable application is the reduction of cyclohexenone to cyclohexanone,
a key reaction in the synthesis of fine chemicals and pharmaceutical
intermediates. In this reaction, the ${\upalpha}$,${\upbeta}$-unsaturated
bond in cyclohexenone is reduced to the corresponding cyclohexanone
with excellent stereoselectivity, highlighting the utility of
ene-reductases for producing optically pure compounds. The versatility
and stability of OYE enzymes, coupled with their ability to perform
reductions under mild conditions, have made them a focal point in
efforts to develop greener and more sustainable chemical processes.
Ongoing research and enzyme engineering have expanded the substrate
scope and improved the efficiency of OYEs, making them a powerful tool
for modern synthetic applications~\cite{42}. 

The \textit{Gs}OYE is a newly identified ene-reductase from
\textit{Galdieria sulphuraria} alga~\cite{41}. It features properties
that fulfill the specifications required for \mbox{industrial} applications,
such as high catalytic activity and robustness (thermostability, wide
range pH optimum, cosolvent tolerance) as well as high-yield
production/purification.

\textit{Gs}OYE was expressed in \textit{E.~coli} with an N-terminal
His-tag. Fe-XCat CarboZym support was then chosen to immobilize the
protein via coordination with iron bound to XCat material. Such
$\mathbi{i}$-\textit{Gs}OYE-Fe-XCat biocatalyst was implemented to
catalyze the reduction of cyclohexenone to cyclohexanone (see
Figure~\ref{fig4} and Supplementary Information 
for protocol details). As described in
Figure~\ref{fig4}a, the biocatalyst was used in batch mode and displays
a conversion ratio ${\sim}$3.5~times higher than the free enzyme from
cycle 1 onwards. It suggests that the enzyme activity can be enhanced
upon immobilization. This may be caused by the fact that the
immobilized state promotes a more catalytically favored conformation
compared to its free form. Reuse of $\mathbi{i}$-\textit{Gs}OYE-Fe-XCat
biocatalyst showed that the enzyme can remain active over five cycles
(with a mean remaining activity around 32\%) resulting in a Total
Turnover Number (TTN) of $5868\,{\pm}\,758$ compared with
$860\,{\pm}\,338$ for the free enzyme.

\begin{figure*}
\includegraphics{fig05}
\vspace*{-5pt}
\caption{\label{fig5}{Results for immobilized B9L0N2}. (a)
Batch mode: conversion ratio and cumulative leaching
([{\textit{buffer}}: 50 mM Tris-HCl ${+}$ 300 mM NaCl ${+}$ 10~wt\%
glycerol; pH 7.5] [{\textit{immobilization conditions}}: 25 mg
Fe-XCat support---10\% (w/w) protein loading---800 rpm; 4~\textdegree
C]; [{\textit{reaction conditions}}: 25 mM pyruvate, 25 mM
racemic ${\upalpha}$-methylbenzylamine, 0.1 mM PLP---25~\textdegree C]); 
(b) Continuous mode: conversion ratio ([{\textit{buffer}}: same
as batch mode] [{\textit{immobilization conditions}}: 70 mg
Fe-XCat support---5\% (w/w) protein loading---0.1 ml/min;
4~\textdegree C]; [{\textit{reaction conditions}}: same as
batch mode; circulation mode---0.1~ml/min---5~hours]).}
\vspace*{-5pt}
\end{figure*}

Continuous mode operation was also tested, where the reaction mixture
was recirculated through a packed column filled with
$\mathbi{i}$-\textit{Gs}OYE-Fe-XCat (Figure~\ref{fig4}b). Over the
course of two hours, \textit{Gs}OYE consistently reduced cyclohexenone,
with steadily increasing conversion rate and TON. It was also shown
that after stopping the process and storing it overnight at
4~\textdegree C, the continuous reaction can be restarted and regained
an efficiency similar to that before storage. The low leaching and
robust activity under continuous conditions position this system as an
effective biocatalyst for scalable processes. It should also be noted
that the leaching measured in batch and continuous mode remains very
low (${&lt;}$1\%), probably thanks to the stable binding provided by the
support/His-tag interaction.
\vspace*{-5pt}

\subsection{Immobilized transaminase for efficient pyruvate conversion}\label{sec33}
\vspace*{-5pt}

Transaminases (TAs) are widely recognized for their role in catalyzing
the transfer of amino groups from amine donors to acceptor molecules,
such as ketones or aldehydes, facilitating the formation of valuable
chiral amines~\cite{43}. These enzymes are particularly attractive for
green chemistry applications in industries such as pharmaceuticals and
agrochemicals, where they are employed for the synthesis of enantiopure
compounds~\cite{44,45}. The demand for such biocatalysts is driven by
their high selectivity, operational stability, and ability to operate
under mild conditions, reducing the need for toxic chemicals and harsh
environments. One example is the transaminase from
\textit{Thermomicrobium roseum} (UniProt accession number B9L0N2),
classified as an ${\upomega}$-transaminase. This class of transaminases
typically operates with both primary amines and \mbox{ketones}, which makes
them versatile tools in \mbox{biotransformations}. These enzymes belong to a
\mbox{family} of enzymes dependent on pyridoxal-5$^{\prime}$-phosphate (PLP)
as a cofactor~\cite{46}. PLP plays a critical role in the catalytic
mechanism, acting as an intermediate carrier of the amino group during
the transamination process. B9L0N2 demonstrates excellent activity in
the asymmetric synthesis of a variety of chiral amines. This enzyme
shows high efficiency in catalyzing the conversion of substrates like
pyruvate and racemic amines, making it a valuable tool in producing
pharmaceutical intermediates~\cite{47}.

The advantages of using B9L0N2 include its ability to perform reactions
under mild conditions and with minimal by-products, as well as its
compatibility with continuous processing, which enhances its potential
industrial applications. Its robustness, combined with low substrate
inhibition and tolerance to organic solvents, makes it highly versatile
for use in different biotechnological settings.

Similarly to \textit{Gs}OYE, B9L0N2 was immobilized on Fe-XCat support
to form a $\mathbi{i}$-B9L0N2-Fe-XCat biocatalyst through
Fe$^{3+}$/His-tag coordination. The transamination of pyruvate in the
presence of racemic ${\upalpha}$-methylbenzylamine was chosen as a model
reaction to evaluate the efficiency of such a biocatalyst both in batch
and continuous mode. The results of these assays are displayed on
Figures~\ref{fig5}a and~\ref{fig5}b, respectively.

The batch test results demonstrate that the enzyme activity remains
highly stable across six \mbox{consecutive} cycles, with a relative residual
activity of approximately 92\% from the first to the sixth cycle. Under
these conditions, the cumulative turnover number over these cycles is
increased by a factor of about 8.75 compared to the enzyme in its free
form, indicating that immobilizing B9L0N2 can reduce enzyme-related
costs proportionally. Given the robust stability of the immobilized
biocatalyst, this factor has the potential to be enhanced further by
extending the number of cycles, underscoring the practicality of
utilizing CarboZym technology for this application.

Additionally, performance in continuous mode (without recirculation)
showed similarly high stability, with a consistent conversion rate
maintained over a five-hour period. Importantly, minimal enzyme
leaching was observed in both batch and continuous setups (${&lt;}$1\%),
supporting the durability of the immobilization. These findings
demonstrate, as a proof of concept, that CarboZym technology offers
significant benefits for the industrial application of transaminases
like B9L0N2, enabling efficient and cost-effective biocatalysis.

\section{Future prospects and industry applications}\label{sec4}
\subsection{Enzyme co-immobilization: mimicking nature's metabolic
efficiency}\label{sec41}

Enzyme co-immobilization represents a sophisticated approach that
bridges the gap between nature's complex metabolic networks and
industrial requirements~\cite{48,49}. This strategy involves the
immobilization of multiple enzymes onto a single support material. It
allows running enzymatic cascades into one-pot reaction systems
creating an artificial microenvironment where sequential enzymatic
transformations can occur efficiently within a single reaction vessel.
By mimicking the intricate choreography of natural metabolic pathways,
this strategy offers a powerful solution to the limitations of
traditional single-enzyme approaches. 

\subsubsection{Fundamental principles and advantages}\label{sec411}

In living cells, metabolic pathways operate through precisely organized
spatial arrangements of enzymes. This natural organization enables
rapid substrate channeling, where intermediates are efficiently
transferred between consecutive enzymes~\cite{50}. Enzyme
co-immobilization seeks to recreate this efficiency in artificial
systems by positioning multiple enzymes on a single support material,
eliminating the need for intermediate isolation or
purification~\cite{51}. 

Traditional single-enzyme approaches face significant limitations when
handling complex multistep reactions. Each enzymatic step typically
requires separate reaction vessels, leading to increased complexity,
cost, and logistical challenges. Moreover, intermediates produced in
one step may degrade before being utilized in subsequent reactions,
resulting in reduced yields and increased purification
demands~\cite{52}. 

Co-immobilization elegantly addresses these challenges by creating a
concentrated microenvironment where enzymes work in concert. The close
proximity of enzymes facilitates direct transfer of intermediates,
minimizing diffusion limitations and byproduct formation. This spatial
arrangement promotes faster reaction rates and shields enzymes from
harsh conditions, enhancing their operational stability and
lifespan~\cite{53}. 

\subsubsection{Design and technical considerations}\label{sec412}

Successfully implementing enzyme co-immobilization requires careful
attention to multiple factors. The choice of support material is
crucial, as it must provide sufficient surface area and appropriate
chemical functionality while maintaining individual enzyme activities.
The material should offer mechanical stability and enable efficient
mass transfer of substrates and products.

Several strategies have emerged for enzyme co-immobilization, each with
distinct advantages. Entrapment within porous matrices, such as
alginate beads or hydrogels, offers a simple and cost-effective
approach but may face diffusion limitations~\cite{54}. Covalent
attachment through chemical bonds provides stable immobilization and
precise control over enzyme positioning but requires careful selection
of coupling chemistry to preserve activity~\cite{55}.

Physical adsorption relies on non-covalent interactions like hydrogen
bonding and electrostatic forces, offering a gentler alternative that
better preserves enzyme activity and allows for enzyme replacement when
needed~\cite{21}. Surface modification techniques enable precise
control over enzyme orientation and density through the creation of
specific binding sites on the support material.

The ratio of different enzymes in the co-immobilized system requires
careful optimization to prevent bottlenecks and ensure smooth reaction
progression~\cite{56}. Unlike natural systems with tightly regulated
enzyme expression, artificial systems need balanced enzyme activities
to match sequential reaction rates.

\subsubsection{Applications across industries}\label{sec413}

The versatility of enzyme co-immobilization has led to its adoption
across various sectors. In pharmaceutical production, co-immobilized
enzymes enable the synthesis of complex drug molecules with high
selectivity and purity, including the production of chiral
intermediates and the biotransformation of drug precursors~\cite{57}.
The food and beverage industry utilizes this technology for producing
modified sweeteners, flavorings, and other additives, while also
enabling the bioconversion of food waste into valuable
products~\cite{58}. In the biofuel sector, co-immobilized enzyme
systems show promise in converting complex biomass into useful
products, particularly in the degradation of cellulosic materials and
the production of biodiesel from vegetable oils~\cite{59}.
Environmental applications include bioremediation of contaminated soils
and water, degradation of toxic pollutants, and the production of
bioplastics from renewable resources~\cite{60}. 

\subsubsection{Current challenges and limitations}\label{sec414}

Despite its potential, enzyme co-immobilization faces several
challenges. The complexity of optimizing multiple parameters
simultaneously makes system design and implementation demanding. Mass
transfer limitations become more significant in co-immobilized enzymes,
requiring careful consideration of substrate and product diffusion
through the support material~\cite{61}. 

The stability of these systems can be compromised if any single enzyme
in the cascade fails, potentially necessitating complete system
replacement. Economic viability often depends on achieving sufficient
operational stability and reusability to justify the initial investment
in expensive support materials and complex immobilization
procedures~\cite{62}. 

\subsubsection{Future perspectives and innovations}\label{sec415}

The future of enzyme co-immobilization lies in developing more
sophisticated systems that better mimic natural metabolic pathways. An
elegant study by Korman et al. designed a homogeneous system comprising
27 enzymes for the conversion of glucose into monoterpenes such as
limonene, pinene and sabinene with high yields~\cite{63}.
Co-immobilization of this entire synthetic biochemistry platform would
probably improve its stability and the total turnover numbers reached
paving the way to challenge cell-based systems.

Advanced materials science and nanotechnology could offer promising
solutions for creating structured support materials with precise
control over enzyme positioning~\cite{64}. 

The field continues to evolve through computational modeling and
artificial intelligence, which could accelerate system optimization by
predicting optimal enzyme arrangements and ratios~\cite{65}. As our
understanding of enzyme interactions and immobilization techniques
advances, co-immobilized systems will likely play an increasingly
important role in enabling sustainable and efficient production
processes across diverse industries~\cite{66}. 

The versatility of CarboZym's immobilization carriers was shown to be
an asset to successfully perform the simultaneous co-immobilization of
up to six different enzymes on the same carrier. We are currently
demonstrating this concept with a simplified enzymatic cascade allowing
the access to terpenoids starting from isopentenol~\cite{67,68,69,70}.
The six overexpressed and purified enzymes were mixed, and XPh was
added to co-immobilize them. This immobilized preparation successfully
catalyzed the conversion of isopentenol to limonene, demonstrating the
successful co-immobilization of the six enzymes and their catalytic
capabilities. This promising study is currently being investigated by
CarboZym and a proof of concept will be published in the near future.

\subsection{Enzymatic cofactor recycling}\label{sec42}

Enzymatic cofactor recycling represents a crucial aspect of industrial
biocatalysis, particularly for reactions involving nicotinamide
cofactors (NADH/NADPH) and adenosine triphosphate (ATP)~\cite{71,72}.
These essential molecules serve as electron carriers and energy sources
in numerous biochemical transformations, but their high cost and
stoichiometric consumption pose significant challenges for industrial
applications.

This overview examines the importance of cofactor recycling systems,
their implementation strategies, and their integration with enzyme
immobilization technologies.

\subsubsection{Fundamental principles}\label{sec421}

Nicotinamide cofactors play vital roles in oxidation--reduction
reactions, serving as electron carriers in numerous enzymatic
transformations. NAD(P)H acts as a reducing agent in many valuable
synthetic processes, including the production of chiral alcohols, amino
acids, and other fine chemicals~\cite{73}. ATP, on the other hand,
functions as an energy carrier, driving thermodynamically unfavorable
reactions and enabling phosphorylation processes essential for many
biotransformations~\cite{74}. 

The economic feasibility of enzymatic processes heavily depends on
efficient cofactor recycling systems. Without recycling, the
stoichiometric use of these expensive cofactors would make most
industrial applications prohibitively expensive. For instance, the cost
of NADH and NADPH can represent a significant portion of the overall
process costs. Similarly, ATP's high cost and inherent instability
necessitate efficient regeneration systems for practical
applications~\cite{75}. 

\subsubsection{Technical approaches to cofactor recycling}\label{sec422}

\paragraph{NAD(P)H recycling}\label{sec4221}

Several strategies have been developed for NAD(P)H regeneration, each
with distinct advantages~\cite{71}. The enzyme-coupled approach employs
secondary enzymes such as formate dehydrogenase, glucose dehydrogenase,
or alcohol dehydrogenase to regenerate the reduced cofactor. These
systems typically use inexpensive sacrificial substrates like formate,
glucose, or isopropanol to drive the regeneration of NAD(P)H.

Substrate-coupled recycling represents an elegant alternative where a
single enzyme catalyzes both the main reaction and cofactor
regeneration. This approach simplifies the reaction system but requires
careful optimization to balance the main reaction and regeneration
rates.

\paragraph{ATP recycling}\label{sec4222}

ATP regeneration systems often utilize phosphoryl transfer enzymes such
as acetate kinase or pyruvate kinase, coupled with high-energy
phosphate donors like acetyl phosphate or phosphoenolpyruvate. These
systems can achieve multiple recycling cycles, though their efficiency
is typically lower than NAD(P)H regeneration systems due to ATP's
complex chemistry and stability issues~\cite{71}. 

\subsubsection{Integration with enzyme immobilization}\label{sec423}

The combination of cofactor recycling with enzyme immobilization
technologies offers promising solutions for industrial
applications~\cite{76}. Co-immobilization of main and recycling enzymes
can create efficient microenvironments where cofactors are rapidly
regenerated and reused. This spatial organization mimics natural
metabolic pathways and can significantly enhance the overall process
efficiency~\cite{77}. 

Various strategies have been developed for immobilizing both enzymes
and cofactors. Cofactor tethering involves the chemical modification of
cofactors to enable their attachment to support materials, while
maintaining their catalytic function. This approach can reduce cofactor
loss but may affect the kinetics of enzymatic reactions due to
diffusion limitations~\cite{78}.

\subsubsection{Applications and industrial impact}\label{sec424}
Cofactor recycling systems have enabled numerous industrial
applications. In the pharmaceutical industry, NAD(P)H-dependent
reactions are crucial for the synthesis of chiral alcohols and amines
which are key building blocks for many active pharmaceutical
ingredients. The integration of efficient recycling systems has made
these processes economically viable at industrial scales~\cite{79}. 

ATP-dependent processes find applications in the production of
phosphorylated compounds, including nucleotides and sugar phosphates.
These molecules serve as important intermediates in various biochemical
pathways and have applications in both pharmaceutical and food
industries.

The combination of cofactor recycling with enzyme immobilization has
led to the development of continuous flow processes, where immobilized
enzyme systems can operate for extended periods with minimal cofactor
loss. These systems offer improved productivity and reduced operational
costs compared to batch processes~\cite{80}. 

\subsubsection{Current challenges}\label{sec425}
Despite significant progress, several challenges remain in the field of
cofactor recycling. The stability of cofactors under industrial
conditions remains a concern, particularly for ATP, which is
susceptible to hydrolysis. The development of more stable cofactor
analogs or protective strategies represents an active area of
research~\cite{81,82}. Mass transfer limitations in immobilized systems
can affect the efficiency of cofactor recycling, particularly when
multiple enzymes and cofactors are involved. The design of carrier
materials with optimal porosity and surface chemistry is crucial for
addressing these limitations~\cite{83}. 

\subsubsection{Future perspectives}\label{sec426}

The successful conversion of isopentenol into limonene with six
co-immobilized enzymes (see Section~\ref{sec415}) also exemplifies the
recycling of ATP. Indeed isopentenol is successively phosphorylated
into isopentenyl phosphate (IP) and isopentenyl diphosphate (IPP)
involving the concomitant consumption of two molecules of ATP yielding
2 $\times$ ADP. In this case, ATP regeneration is catalyzed by an acetate
kinase (one of the six co-immobilized enzymes) using acetyl phosphate
as a phosphate donor~\cite{8}. 

The continued development of innovative solutions that address current
limitations while maintaining economic viability will shape the future
of this field, enabling more sustainable and efficient production
processes across various industries.

\subsection{Scaling up hydrothermal carbonization: from laboratory to
industrial reality}\label{sec43}

The CarboZym technology is based on the hydrothermal carbonization, or
more accurately the solvothermal carbonization, of sugar in the
presence of phenolic compounds. Hydrothermal carbonization (HTC) is a
thermochemical process that transforms organic matter into valuable
carbon-based materials under subcritical conditions, i.e., at
relatively high temperature and pressure~\cite{84}. HTC has
demonstrated its efficacy at the laboratory scale and holds immense
potential for sustainable material production~\cite{84,85}. However,
translating this technology to industrial scale presents a unique set
of challenges and opportunities that have been partially addressed in
the literature for wet biomass wastes~\cite{86} and sewage
sludges~\cite{87}. Several companies have developed HTC plants at pilot
and industrial scales, such as C-Green (OxyPower HTC{\texttrademark}
plant in Finland), TerraNova Energy, Ingelia, CarboREM \mbox{(CarboREM}
\mbox{C-700}
Plant \mbox{installed} in Italy), and HTC-Cycle. Technical, economic, and
environmental considerations are crucial for successfully scaling up
HTC processes for porous carbon-based material production~\cite{84}.
Even though CarboZym technology involves partially purified precursors
that are soluble and therefore easier to process, the scale-up remains
challenging and necessitates a meticulous approach to reactor design
and operational parameters. \looseness=1

\subsubsection{Technical hurdles and engineering solutions}\label{sec431}

\paragraph{Reactor design and operation: navigating heat and pressure}

As reactor volume increases, the efficient transfer of heat becomes
paramount. Maintaining uniform temperature distribution within the
reaction medium is critical for consistent product quality and requires
careful consideration of heating systems and mixing mechanisms.
Efficient external jackets, internal heating elements, and robust
mixing systems, such as impellers or recirculation loops, are essential
for achieving homogeneous heating. In a recent industry perspective on
zeolite manufacturing, Maurer and Parvulescu from BASF discussed heat
distribution heterogeneities within high volume synthesis reactors
(10--20~m$^{3}$)~\cite{88}. A computational fluid analysis showed that
even for well-stirred and heated reactor gradients, mixing flows and
temperature distributions could occur during the hydrothermal
treatment~\cite{88}.

Pressure management is another crucial aspect. HTC processes typically
operate under autogenous pressure, ranging from 10 to 40 bar, demanding
robust reactor construction materials capable of withstanding these
conditions. Corrosion resistance is equally important, given the acidic
nature of the reaction medium (pH is usually below 3 at the end of
reaction). Selecting materials like stainless steel alloys or
specialized corrosion-resistant coatings is vital for reactor longevity
and safety. However, scaling up the synthesis of porous materials under
these conditions is not insurmountable; the commercial scale production
of zeolites~\cite{88,89}, in particular Zeolite A (LTA), is a proof.
Large-volume reactors of 10--20~m$^{3}$ have been reported for
manufacturing zeolites at commercial scale using conventional batch
technologies. Process intensification via continuous flow technology is
currently developed by Arkema at pilot scale~\cite{90}. 

\paragraph{Process control and monitoring: ensuring consistency and quality}

Maintaining consistent product quality at industrial scale requires
sophisticated control systems. Continuous monitoring of key parameters,
including temperature, pressure, residence time, and slurry
concentration, is essential. Implementing in-line sensors and automated
control systems allows for real-time adjustments, minimizing deviations
from optimal operating conditions and ensuring product uniformity. Data
acquisition and analysis can provide valuable insights into process
performance, enabling continuous improvement and optimization.

\paragraph{Feed handling, product recovery, washing and drying:
streamlining the process}

Scaling up feed handling systems presents unique challenges. Solutions
have been proposed for the continuous flow synthesis of zeolites with
in-line mixing of the precursors and in-line seeding of the synthesis
gel solution~\cite{90}. Efficient downstream processing is also
essential. As mentioned above (Section~\ref{sec22}), two crucial downstream
processing steps are required to handle wet gels and slurries after
HTC, e.g., solvent exchange through intensive washing, and drying.
These steps will be inspired by downstream processing of hydrochar and
zeolite slurries adopted in pilot plants, but also by other cryo- and
aerogel technologies such as Starbon(R), a spin out from the University
of York's Green Chemistry Centre of Excellence~\cite{91}. Besides,
efficient solid--liquid separation techniques are essential for
recovering the HTC-produced carbon-based material. Filtration methods,
such as membrane filtration or pressure filtration, may be employed
depending on the desired particle size and purity. 

\subsubsection{Economic viability: balancing costs and returns}\label{sec432}

The economic viability of scaled-up HTC processes hinges on several key
factors.

\paragraph{Raw material supply: securing a steady stream}

Securing a consistent supply of suitable biomass feedstock at
competitive prices is paramount. The scale-up strategy must consider
feedstock \mbox{availability}, transportation costs, and potential seasonal
variations in supply. Diversifying feedstock sources and establishing
strong partnerships with biomass suppliers can mitigate supply chain
risks.

\paragraph{Operating costs: optimizing efficiency and minimizing expenses}

Energy consumption, particularly for heating and potential activation
processes, significantly impacts operating costs. Implementing heat
recovery systems, optimizing process parameters, and exploring
alternative energy sources, such as biomass-derived fuels, can
contribute to improved energy efficiency and reduced operational costs.

\paragraph{Capital investment: weighing the initial outlay}

The initial capital investment required for HTC facilities is
substantial. Careful financial planning, thorough market analysis, and
a robust business plan are essential for assessing the return on
investment.

Modular design approaches, where production capacity can be
incrementally increased, offer flexibility in adapting to changing
market demands and minimizing initial capital requirements.

\subsubsection{Overcoming challenges: ensuring sustainability and consistency}

\paragraph{Product consistency: maintaining quality at scale}

Maintaining consistent product quality across large production volumes
is a key challenge. Implementing robust quality control systems,
standardized operating procedures, and rigorous testing protocols are
essential for ensuring that the HTC-produced carbon-based materials
meet the required specifications.

\paragraph{Environmental impact: minimizing the footprint}

Managing process emissions and wastewater treatment at industrial scale
requires careful consideration. The development of closed-loop systems
for water recycling and emission control becomes increasingly
important. Implementing strategies for capturing and utilizing process
emissions, such as carbon dioxide sequestration or biogas production,
can further enhance the environmental sustainability of HTC processes.

\subsubsection{Future prospects: innovation driving advancement}

The future of HTC scale-up lies in continuous innovation and
technological advancements.

\paragraph{Advanced process control: harnessing the power of data}

The integration of artificial intelligence and machine learning can
enable more sophisticated process control and optimization, leading to
improved product consistency, reduced operating costs, and enhanced
process efficiency.

{\vspace*{-1pt}}

\paragraph{Sustainable processing: embracing circular economy
principles}

Integrating renewable energy sources, such as solar or wind power, can
reduce the carbon footprint of HTC processes. Implementing circular
economy principles, such as utilizing the HTC-derived soluble byproduct
as biofuels or platform molecules for biorefinery schemes, can further
enhance the sustainability and economic viability of HTC.\looseness=-1

{\vspace*{-2pt}}

\subsubsection{Conclusion: a promising path forward}

{\vspace*{-1pt}}

Scaling up HTC for porous carbon-based material production presents a
complex yet promising opportunity for industrial development. Success
requires a multifaceted approach, addressing technical, economic, and
environmental considerations. By embracing innovation, sustainability,
and a commitment to responsible development, HTC has the potential to
become a key technology for producing high-quality carbon-based
materials, contributing to a more sustainable and resource-efficient
future~\cite{84,85}. CarboZym has already reached key milestones,
producing several hundred grams of support materials per week at
laboratory scale with a high degree of homogeneity and reproducibility.

{\vspace*{-2pt}}

\section{Conclusion}\label{sec5}

{\vspace*{-2pt}}

This report highlights the transformative potential of CarboZym
technology through three compelling case studies. CarboZym's
carbon-based, biomass-derived carriers offer a unique advantage in
biomanufacturing by minimizing reliance on petrochemicals and aligning
with the industry's growing demand for low-carbon solutions.

The simple immobilization procedure, demonstrated with industrially
relevant enzymes (CALB, \textit{Gs}OYE, and ${\upomega}$-TA B9L0N2),
proved quick and highly effective, achieving 95--100\% immobilization
within approximately two hours. This success underscores the
versatility of CarboZym's applicability in green chemistry and
sustainable biocatalysis. Each \mbox{immobilized} enzyme exhibited distinct
catalytic \mbox{enhancements,} showcasing the platform's adaptability to
diverse enzymatic systems.

Immobilized CALB demonstrated exceptional stability and reusability,
maintaining industrially relevant conversion rates over multiple cycles
with minimal enzyme leaching. \textit{Gs}OYE exhibited excellent
efficiency and stability in both batch and continuous flow systems,
achieving excellent turnover numbers in continuous mode. Immobilized
${\upomega}$-TA B9L0N2 achieved significant cost reductions through
repeated use, retaining nearly full activity.

By combining high catalytic performance, low leaching, enhanced
reusability, and a sustainable footprint, CarboZym's immobilization
platform addresses key challenges facing biomanufacturing.

The broader implications of this technology extend far beyond
individual applications. As the biomanufacturing industry strives for
greater sustainability and efficiency, CarboZym emerges as a crucial
enabler of this transition. Successful scale-up and adoption of
CarboZym-based systems can significantly reduce costs, accelerating the
shift towards greener, more circular industrial practices.

In conclusion, the CarboZym technology represents a transformative
approach to enzyme immobilization, poised to reshape the future of
sustainable biomanufacturing. By seamlessly integrating
high-performance catalysis, operational robustness, and environmental
responsibility, CarboZym paves the way for a more economically and
ecologically viable industrial biotechnology landscape, unlocking new
avenues for innovation and growth.

{\vspace*{-2pt}}

\section*{Declaration of interests}

{\vspace*{-2pt}}

JD, NB and CM are declared inventors on patents WO2024153593A1,
WO2024153594A1 used by CarboZym.  JD, NB and QH are cofounders of the
CarboZym SAS.

{\vspace*{-2pt}}

\section*{Funding}

{\vspace*{-2pt}}

This project was supported by Univ.\ Montpellier (LabMUSE Chimie 2021),
R\'{e}gion Occitanie (Pre-maturation 2021) and SATT AxLR
(Pre-maturation 2021, Maturation 2023).

\back{}

{\vspace*{-2pt}}

\section*{Supplementary data}

{\vspace*{-2pt}}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-397-suppl.pdf}}

\printbibliography
\refinput{crchim20240966-reference.tex}

\end{document}
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