<html><head><meta name="color-scheme" content="light dark"></head><body><pre style="word-wrap: break-word; white-space: pre-wrap;">\makeatletter
\@ifundefined{HCode}
{\documentclass[screen,CRCHIM,Unicode,biblatex,published]{cedram}
\addbibresource{crchim20240938.bib}
\newenvironment{noXML}{}{}
\def\tsup#1{$^{{#1}}$}
\def\tsub#1{$_{{#1}}$}
\def\thead{\noalign{\relax}\hline}
\def\tbody{\noalign{\relax}}
\def\endthead{\noalign{\relax}\hline}
\RequirePackage{etoolbox}
\usepackage{upgreek}
\let\rmgamma\upgamma
\let\rmalpha\upalpha
\let\rmbeta\upbeta
\def\jobid{crchim20240938}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\newcounter{runlevel}
\let\MakeYrStrItalic\relax
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\def\back#1{}
\def\0{\phantom{0}}
\newenvironment{inftab}{}{}
\usepackage{multirow} 
\def\nrow#1{\@tempcnta #1\relax%
\advance\@tempcnta by 1\relax%
\xdef\lenrow{\the\@tempcnta}}
\def\morerows#1#2{\nrow{#1}\multirow{\lenrow}{*}{#2}}
\def\ubreak{{\unskip\break}}
\def\hbreak{{-\break}}
\DOI{10.5802/crchim.405}
\datereceived{2024-11-13}
\daterevised{2025-03-06}
\dateaccepted{2025-05-20}
\ItHasTeXPublished
}
{\documentclass[crchim]{article}
\def\CDRdoi{10.5802/crchim.405}
\let\newline\break
\let\ubreak\relax
\let\hbreak\relax
\makeatletter
}
\makeatother

\usepackage{upgreek}

\dateposted{2025-06-20}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Biocatalysis, a great toolbox for the upgrading of biomass}

\alttitle{La biocatalyse, une bo\^{i}te \`{a} outils formidable pour la
valorisation de la biomasse}

\author{\firstname{Louis Michel Marie} \lastname{Mouterde}\CDRorcid{0000-0002-4096-1629}\IsCorresp\IsEqualContrib}
\address{URD Agro-Biotechnologies Industrielles (ABI), 
CEBB, AgroParisTech, 51110, Pomacle, France}
\email[L. M. M. Mouterde]{Louis.mouterde@agroparistech.fr}

\author{\firstname{Florent} \lastname{Allais}\CDRorcid{0000-0003-4132-6210}\IsCorresp\IsEqualContrib}
\addressSameAs{1}{URD Agro-Biotechnologies Industrielles (ABI), 
CEBB, AgroParisTech, 51110, Pomacle, France}
\email[F. Allais]{florent.allais@agroparistech.fr}

\thanks{R\'{e}gion Grand Est, Conseil D\'{e}partemental de la Marne, Grand Reims}

\begin{abstract}
The development and application of biocatalysis have evolved
significantly, from the classical use of commercial enzymes to the
production, modification and implementation of non-commercially
available enzymes, driving advancements in sustainable bioprocesses for
bio-based chemical and material production. We have focused on
combining chemistry, biocatalysis and process intensification, to
create efficient (chemo)enzymatic routes capable of transforming
renewable raw materials into valuable industrial products. The
portfolio of biocatalysts has been expanded through the integration of
biocatalysis and green chemistry principles, facilitating innovative,
eco-friendly alternatives to traditional petrochemical processes and
supporting the transition towards a circular bioeconomy.
\end{abstract}

\keywords{\kwd{Green chemistry}
\kwd{Biocatalysis}
\kwd{Molecular biology}
\kwd{Enzymes}
\kwd{Biomass}
\kwd{Bio-based molecules}}

\altkeywords{\kwd{Chimie verte}
\kwd{Biocatalyse}
\kwd{Biologie mol\'{e}culaire}
\kwd{Enzymes}
\kwd{Biomasse}
\kwd{Mol\'{e}cules biosourc\'{e}es}}

\begin{altabstract}
Le d\'{e}veloppement et l'application de la biocatalyse ont
consid\'{e}rablement \'{e}volu\'{e}, passant de l'utilisation classique
d'enzymes commerciales \`{a} la production, la modification et la mise
en \oe{}uvre d'enzymes non disponibles dans le commerce. Cette
avanc\'{e}e a permis de faire progresser les bioproc\'{e}d\'{e}s
durables pour la production de produits chimiques et de mat\'{e}riaux
d'origine v\'{e}g\'{e}tale. Nous nous sommes concentr\'{e}s sur la
combinaison de la chimie, de la biocatalyse et de l'intensification des
processus, afin de cr\'{e}er des voies (chimio)enzymatiques efficaces
capables de transformer les mati\`{e}res premi\`{e}res renouvelables en
produits industriels de valeur. Le portefeuille de biocatalyseurs a
\'{e}t\'{e} \'{e}largi gr\^{a}ce \`{a} l'int\'{e}gration de la
biocatalyse et des principes de la chimie verte, offrant des
alternatives innovantes et respectueuses de l'environnement aux
processus p\'{e}trochimiques traditionnels et favorisant la transition
vers une bio\'{e}conomie circulaire.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}
URD ABI (Agro-Industrial Biotechnologies Research and Development 
Unit)~\cite{1}, a research unit within AgroParisTech~\cite{2}, is
located in the European Center for Biotechnology and Bioeconomy (CEBB)
at the heart of the Pomacle--Bazancourt  biorefinery~\cite{3}. URD ABI
drives innovation and its transfer to industrialization, covering the
Technology Readiness Level (TRL) scale from 1 to 4. More precisely, URD
ABI aims to transform plant and agricultural materials, as well as
agro-industrial by-products, into sustainable industrial solutions,
aligning with both the bioeconomy concept and several of the United
Nations Sustainable Development Goals  (UNSDG)~\cite{4}.

One pillar of URD ABI, biocatalysis has been at the heart of many
flagship projects since the unit was founded. Initially limited to the
use of commercial enzymes and liquid fermentation, URD ABI's expertise
in the field of biotechnology has rapidly expanded to molecular
biology, and enzymology.

In this mini-review, we will use a few examples to illustrate how
biocatalysis has evolved within URD ABI and how it is now positioned as
a major asset in the development of sustainable access routes to
molecules and materials with (very) high added value.

\begin{scheme*}
\vspace*{2pt}
\includegraphics{sc01}
\vspace*{.5pc}
\caption{\label{sch1}Esterase-mediated production of ferulic acid and
sinapic acid from wheat bran and sugar beet pulp, and mustard and
rapeseed cakes, respectively.}
\end{scheme*}

\section{Discussion}\label{sec2}

\subsection{Episode 1: First steps in biocatalysis}\label{sec2.1}
Initially, biocatalysis activities at URD ABI were limited to the use
of commercially available enzymes. To stand out from the stiff
competition in this field, we focused on the use of lipases and
laccases for the valorization of phenolic molecules, e.g.,
\mbox{$p$-hydroxycinnamic} acids and the 
\mbox{corresponding} benzaldehydes 
(Figure~\ref{fig1}), which until then had remained relatively
unexplored.

\begin{figure}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-3pt}
\caption{\label{fig1}Naturally occurring $p$-hydroxy{\hbreak}cinnamic
acids and corresponding benzaldehydes.}
\vspace*{-5pt}
\end{figure}

At first, the project focused on the production of new bis- and
tris-phenolic molecules to replace bisphenol A, a petrochemical
criticized for its toxicity (endocrine disruption) and allergenicity.
Starting directly from the $p$-hydroxycinnamic acids that can be
readily obtained from local biomasses (ferulic acid in wheat bran and
sinapic acid in rapeseed meal or mustard bran) through commercially
available esterase-mediated hydrolysis
(Scheme~\ref{sch1})~\cite{5a,5b,5c}, we have developed and optimized
two chemo-enzymatic synthetic routes leading to a new family of
bis/trisphenols and syringaresinol, respectively 
\mbox{(Figure~\ref{fig2}).}

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig02}
\vspace*{-5pt}
\caption{\label{fig2}Ferulic-acid-based bis- and trisphenols and
sinapic-acid-derived syringaresinol.}
\vspace*{-5pt}
\end{figure*}

The synthesis of ferulic-acid-based bis- and trisphenols and
sinapic-acid-based bisphenol are based on a one-pot two-step Fisher
esterification reaction and pallado-catalyzed hydrogenation of ferulic
or sinapic acids, followed by transesterification of the resulting
saturated ester by diols (1,3-propanediol, 1,4-butanediol, isosorbide)
and triol (glycerol) by  using \textit{Candida antarctica} type B
lipase immobilized on polystyrene beads (aka Novozyme 435, CAL-B) 
(Scheme~\ref{sch2})~\cite{6}. 

\begin{scheme*}
\includegraphics{sc02}
\vspace*{.6pc}
\caption{\label{sch2}Chemo-enzymatic synthesis of ferulic-acid-derived
bis- and trisphenolic esters.}
\end{scheme*}

Coupled with a membrane purification process~\cite{7}, this
solvent-free biocatalysis, carried out under reduced pressure, enables
the production of bis- and trisphenols with high yields  (90--95\%) and
purity (98\%), and has been validated on a kilogram scale. It is
noteworthy to mention that this strategy has also been successfully
applied to the production of bisphenol  amides~\cite{8}. 

The second chemo-enzymatic synthetic route developed is based on a
strategy fairly similar to the previous one. Sinapic acid was
esterified and then reduced by DIBAL-H to afford the corresponding
allyl alcohol. The latter was then subjected to a laccase  from
\textit{Trametes versicolor}, in a buffer solution, to obtain
syringaresinol in 93\% yield  (Scheme~\ref{sch3})~\cite{9}. It is
important to note here that the high selectivity of the 
reaction---obtained by using a design of  experiment---allows the
almost exclusive production of syringaresinol ($\rmbeta$--$\rmbeta$
dimer) and the absence of the $\rmbeta$-O-4 dimer, which is generally
the major product of this reaction. 

\begin{scheme*}
\includegraphics{sc03}
\vspace*{.6pc}
\caption{\label{sch3}Chemo-enzymatic synthesis of syringaresinol from
sinapic acid.}
\end{scheme*}

As the original aim was to propose biosourced, non-toxic alternatives
to bisphenol A, the study of the toxicity of the aforementioned
compounds was realized in collaboration with INSERM Montpellier and
showed that they did not exhibit any activity, regardless of their
concentration  (Figure~\ref{fig3})~\cite{10}. 

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Endocrine disruption activity of bisphenol A and
bio-based bis- and trisphenols.}
\end{figure*}

Moreover, possessing free phenol moieties, all compounds exhibit
valuable antioxidant 
\mbox{activities,} both in solution or in 
plastics~\cite{11,12}. Interestingly, the antioxidant activity was
further enhanced through a highly selective laccase-mediated
oligomerization of ferulic-acid-based bisphenols 
(Scheme~\ref{sch4})~\cite{13}. 

\begin{scheme*}
\includegraphics{sc04}
\vspace*{.5pc}
\caption{\label{sch4}Laccase-mediated regioselective oligomerization of
bisphenols.}
\end{scheme*}

Subsequently, in collaboration with INRAE, Rensselaer Polytechnic
Institute, and ICGM, we demonstrated that, of all the phenolic
compounds developed, syringaresinol led to epoxy-amine resins with
thermo-mechanical properties similar to those of bisphenol A-based 
ones~\cite{14}, whereas the bisphenolic esters provided degradable
epoxy-amine  resins~\cite{10,15}. It is worth mentioning that these
bisphenols were also used as monomers for the production of bio-based
non-isocyanate polyurethanes  (NIPUs)~\cite{16,17}. High added-value
applications have been developed from these molecules. For example,
butane-1,4-diyl bis(3-(4-hydroxy-3-methoxyphenyl){\ubreak}propanoate) 
(BDF), the bisphenol derived from ferulic acid and 1,4-butanediol, has
been used to develop  (i)~new polylactic acid (PLA)/BDF blends with
shape-memory elastomer  properties~\cite{18,19},  (ii)~self-healing
materials~\cite{20}, and  (iii)~reversible adhesives~\cite{21}.

More recently, still in the field of phenolic compounds, biocatalysis
has been put to good use in a project dedicated to the production of
naturally\unskip\break 
occurring sinapoyl malate  (Scheme~\ref{sch5}), which is
proving a multifunctional phenolic compound with UV-filtering,
antioxidant and anti-tyrosinase properties for the cosmetics and
biocontrol  sectors~\cite{22}.

\begin{scheme*}
\includegraphics{sc05}
\vspace*{.5pc}
\caption{\label{sch5}Sustainable three-step synthesis of sinapoyl
malate.}
\vspace*{.7pc}
\end{scheme*}

Although highly active, sinapoyl malate was difficult to formulate due
to  the $p$-hydroxycinnamic acid ester core and the two acid moieties.
To overcome this problem, one solution was to add hydrophobic moieties
to the two malic esters. However, such a selective
(trans)esterification of these malic acids/esters, without affecting
the $\rmalpha$,$\rmbeta$-unsaturated ester, was not conceivable by
purely chemical means. Fortunately, by exploiting the inability of
CAL-B to  transesterify $p$-hydroxycinnamic acids, we were able to
graft the fatty chains in a completely regioselective manner to produce
new and more easily formulated analogs  (Scheme~\ref{sch6})~\cite{23}.

\begin{scheme*}
\includegraphics{sc06}
\vspace*{.5pc}
\caption{\label{sch6}Lipase-mediated regioselective transesterification
toward fatty esters of sinapoyl malate.}
\end{scheme*}

CAL-B has also been used for the valorization of levoglucosenone (LGO),
a chiral molecule with high added value obtained through the pyrolysis
of  cellulose~\cite{24}. Because of its chirality, 
$\rmalpha$,$\rmbeta$-unsaturated ketone and ketal, LGO is an ideal 
\mbox{platform} \mbox{molecule} for organic chemists. Among the many
chiral molecules that can be synthesized from  LGO,  
$(S)$-$\rmgamma$-hydroxymethyl-$\rmalpha$,$\rmbeta$-butenolide (HBO)
and its reduced derivative 
($(S)$-$\rmgamma$-hydroxymethyl-$\rmgamma$-butyrolactone, 2H-HBO) are
probably the ones that open up the widest range of possibilities 
(Scheme~\ref{sch7}). 

\begin{scheme}
\includegraphics{sc07}
\vspace*{.7pc}
\caption{\label{sch7}LGO, a valuable chiral chemical platform.}
\vspace*{-.5pc}
\end{scheme}

These chiral butyrolactone/butenolide can be used to produce 
drugs~\cite{25}, aromas~\cite{26},  polymers~\cite{27,29} etc. Until
the URD ABI studied its production from LGO, HBO was obtained by
Baeyer-Villiger oxidation of LGO, either in the presence of 
peracids~\cite{30} or in the presence of zeolites~\cite{31} 
(Scheme~\ref{sch8}). In both cases, although effective in terms of
yield, these two chemical processes are nevertheless dangerous and
toxic for humans as well as for the environment due to the use of toxic
and/or explosive reagents and toxic solvents.

\begin{scheme}
\includegraphics{sc08}
\vspace*{.7pc}
\caption{\label{sch8}Hazardous and toxic chemical synthetic routes
toward LGO.}
\vspace*{-.5pc}
\end{scheme}

\begin{scheme*}
\includegraphics{sc09}
\vspace*{.5pc}
\caption{\label{sch9}Lipase-mediated Baeyer-Villiger oxidation of
unsaturated and saturated LGO into HBO and 2H-HBO, respectively.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc10}
\vspace*{.5pc}
\caption{\label{sch10}Bioconversion of Cyrene{\texttrademark} into
2H-HBO.}
\end{scheme*}

To overcome these limitations, we designed and optimized a sustainable
alternative chemical pathway based on the use of CAL-B and hydrogen
\mbox{peroxide} in ethyl acetate (AcOEt) to produce in situ peracetic acid,
which oxidizes LGO to HBO in high yields without the need for harsh
oxidizing agents in large excess or hazardous petroleum-based organic
solvents  (Scheme~\ref{sch9})~\cite{32}.  It is noteworthy to mention
that, to recycle CAL-B, HEPES buffer can be\unskip\break 
used.

As the above examples show, the implementation of biocatalytic
reactions using commercially available enzymes has been a key element
in the strategy of URD ABI for the production of high added-value
molecules and materials using sustainable processes. However, due to
the relatively limited number of enzymes on the market, the high
potential of biocatalysis could not be fully exploited within URD ABI.
Aware of these limitations, it was decided to acquire the capacity to
produce enzymes on site and thus go even further in terms of
innovation.

\subsection{Episode 2: Fully harnessing the potential of molecular
biology and biocatalysis}\label{sec2.2}
With these new capacities, and in collaboration with the University of
Florida, we first explored another biocatalytic strategy for the
conversion of LGO into HBO via cyclohexanone monooxygenase (CHMO). This
strategy has the advantage of  (i)~using oxygen from air as oxidative
species and therefore significantly improving the system in terms of
safety compared to H$_{2}$O$_{2}$ and,  (ii)~not implementing organic
solvent such as AcOEt. However, the use of the CHMO collection from
Stewart Lab did not lead to positive results and no activity was
observed. However, using  Cyrene{\texttrademark} as substrate allowed
us to observe the formation of 2H-HBO. We then explored the
biodiversity using the basic local alignment search tool (BLAST) and
the DNA sequence coding for the CHMO  from \textit{Acinetobacter} sp.\ 
NCIB 9871 as the parent sequence in an attempt to find new CHMOs. A hit
from \textit{Pseudomonas aeruginosa} strain Pa1242 was identified with
a query cover and identity percentage of 97 and 67\% respectively. As
CHMOs are NADPH-dependent, a quite expensive cofactor, we further
implemented the system in whole cells to take advantage of the cells'
capacity to regenerate NADPH by themselves  (Scheme~\ref{sch10}). Doing
so allowed us to reach a concentration of 20 g/L (160 mM) over 48 h or
a consumption rate of $401\pm36$~mg/L/h~\cite{33,34}. 

\begin{scheme*}
\includegraphics{sc11}
\vspace*{.5pc}
\caption{\label{sch11}Biocatalytic transformation of LGO into
Cyrene{\texttrademark}.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc12}
\vspace*{.5pc}
\caption{\label{sch12}Simplification of the reaction medium using
apyrase.}
\end{scheme*}

As it was previously discussed in this review, it is possible to access
aromas from LGO, such as the flavoring agent dairy lactone (butter,
peach, or fruity aroma depending on the concentration 
used)~\cite{26,35}. The transfer from a classical organic step toward a
biocatalytic one is of particular interest in such a pathway. Indeed,
naturality is progressively becoming a requested standard for almost
all industries nowadays and it is particularly the case for the food
industry. The implementation of a classical organic synthesis step in a
pathway causes the natural character of the targeted molecule to be
lost. With this first proof of feasibility, we decided to go further in
trying to substitute each step by a biocatalytic system in order to
access natural dairy lactone. Therefore, we explored the possibility of
transforming LGO into  Cyrene{\texttrademark} by old yellow enzymes
(OYEs) as it could add a second biocatalytic step toward dairy lactone
and provide  Cyrene{\texttrademark} through a method that does not use
transition metals such as palladium. Indeed, some industries that could
use  Cyrene{\texttrademark} as substitute for DMSO, DMF or NMP are
required to have very small traces of transition metal, parts per
billion, and a biocatalytic pathway would be able to provide such a
grade. The OYE 2.6  from \textit{Pichia stipitis} was chosen as it
displayed the best activity from the set of OYEs  tested~\cite{37,38}.
Contrary to the transformation of  Cyrene{\texttrademark} into 2H-HBO,
the whole-cell strategy for scaling up the process could not be applied
because of the high toxicity of LGO toward cells. Therefore, a
regeneration system of NADPH has been implemented using glucose
dehydrogenase and glucose as the sacrificial agent. The process was
scaled up to five liters with a final concentration of 250 mM 
(Scheme~\ref{sch11}). 

\begin{scheme*}
\includegraphics{sc13}
\vspace*{.5pc}
\caption{\label{sch13}Biocatalytic production of pantethine diphosphate
using CNC-ATP.}
\end{scheme*}

Although the system was proven to be efficient, its cost remains
relatively high, mostly due to the use of  NADP$^{+}$, which represents
more than 70\% of raw material cost. Therefore, we looked into a
strategy that could allow us to reduce the production cost. We
identified that the purification steps of NADP$^{+}$ production
represents the majority of the cost as it is usually performed through
ion exchange chromatography followed by a desalting step using
activated charcoal. In order to overcome such drawbacks, we developed
and patented a membrane filtration strategy that allows purifying high
molecular weight adenosine-based cofactors in a 3-to-1 process that
enables  (i)~purifying,  (ii)~desalting, and  (iii)~recycling the
by-products. The proof of feasibility was made on coenzyme A, FAD, NAD,
and  NADP$^{+}$~\cite{39,40}. In the case of NADP$^{+}$, we added a
final step involving an apyrase from potatoes in order to simplify the
profile of by-products  (Scheme~\ref{sch12}), allowing us to reach
higher purity and  productivity~\cite{41}.

Another approach investigated to mitigate coenzyme-associated expenses
was the immobilization of the coenzymes on cellulose nanocrystals
(CNCs). We used ATP as a case study and were able to graft it by a
click chemistry approach onto CNCs. We then used the resulting material
in a biocatalysis involving a pantothenate kinase  from 
\textit{E.~coli} for the production of pantethine diphosphate and
showed that the biocatalysis was occurring as observed when using 
\textit{free} ATP  (Scheme~\ref{sch13}). It must be noted that both
mono and dephosphorylated products were observed. Such results open up
real perspectives for cofactor  immobilization~\cite{42}.

Unlocking the access to coenzymes is crucial for biocatalysis as it can
give access to a very large variety of biocatalytic activities. 

\section{Conclusion}\label{sc3}
By integrating chemistry and biocatalysis, a powerful approach has been
implemented for developing sustainable processes aimed at producing
bio-based chemicals and materials. This interdisciplinary synergy
leverages the precision of chemical methods alongside the selectivity
and eco-friendliness of biological catalysts, such as enzymes and
microorganisms. Biocatalysts enable highly specific transformations
under mild reaction conditions, often reducing energy requirements and
minimizing the need for toxic solvents or harsh chemicals. When
combined with advanced chemical methods, biocatalysis can enhance
reaction efficiency, yield, and selectivity, opening new pathways for
the valorization of renewable biomass into high-value chemicals,
polymers, and advanced materials. Key advancements in enzyme
engineering, metabolic pathway optimization, and hybrid chemo-enzymatic
processes have expanded the repertoire of bio-based products that can
be produced sustainably, offering promising alternatives to
petrochemical-derived products. This combined approach supports
circular economy principles by using renewable feedstocks and designing
biodegradable end-products, ultimately reducing environmental impact
and reliance on limited 
\mbox{resources.}

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}
Authors would like to thank the R\'{e}gion Grand Est, the Conseil
D\'{e}partemental de la Marne and the Grand Reims for their financial
support.

\back{}

\printbibliography
\refinput{crchim20240938-reference.tex}

\end{document}
</pre></body></html>