Citation: Heinks, T.; Merz, L.M.;
Liedtke, J.; Höhne, M.;
van Langen, L.M.; Bornscheuer, U.T.;
Fischer von Mollard, G.; Berglund, P.
Biosynthesis of Furfurylamines in
Batch and Continuous Flow by
Immobilized Amine Transaminases.
Catalysts 2023,13, 875. https://
doi.org/10.3390/catal13050875
Academic Editor: Rong Shi
Received: 31 March 2023
Revised: 29 April 2023
Accepted: 9 May 2023
Published: 11 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
catalysts
Article
Biosynthesis of Furfurylamines in Batch and Continuous Flow
by Immobilized Amine Transaminases
Tobias Heinks 1,† , Luisa M. Merz 2,3,† , Jan Liedtke 1, Matthias Höhne 4, Luuk M. van Langen 3,
Uwe T. Bornscheuer 5, Gabriele Fischer von Mollard 1and Per Berglund 2,*
1Faculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany;
2Department of Industrial Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center,
3ViaZym B.V., Molengraaffsingel 10, 2629JD Delft, The Netherlands; [email protected]
4
Biocatalysis/Biological Chemistry, Technical University Berlin, Müller-Breslau-Str.10, 10623 Berlin, Germany;
5Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald,
Felix Hausdorff-Str. 4, 17487 Greifswald, Germany; uwe.bornscheuer@uni-greifswald.de
*Correspondence: [email protected]
† These authors contributed equally to this work.
Abstract:
Building blocks with amine functionality are crucial in the chemical industry. Biocatalytic
syntheses and chemicals derived from renewable resources are increasingly desired to achieve
sustainable production of these amines. As a result, renewable materials such as furfurals, especially
furfurylamines like 5-(hydroxymethyl)furfurylamine (HMFA) and 2,5-di(aminomethyl)furan (DAF),
are gaining increasing attention. In this study, we identified four different amine transaminases (ATAs)
that catalyze the reductive amination of 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran
(DFF). We successfully immobilized these ATAs on glutaraldehyde-functionalized amine beads using
multiple binding and on amine beads by site-selective binding of the unique C
α
-formylglycine
within an aldehyde tag. All immobilized ATAs were efficiently reused in five repetitive cycles of
reductive amination of HMF with alanine as co-substrate, while the ATA from Silicibacter pomeroyi
(ATA-Spo) also exhibited high stability for reuse when isopropylamine was used as an amine donor.
Additionally, immobilized ATA-Spo yielded high conversion in the batch syntheses of HMFA and
DAF using alanine (87% and 87%, respectively) or isopropylamine (99% and 98%, respectively) as
amine donors. We further demonstrated that ATA-Spo was effective for the reductive amination
of HMF with alanine or isopropylamine in continuous-flow catalysis with high conversion up to
12 days (48% and 41%, respectively).
Keywords:
amine transaminase; biocatalysis; DFF; flow synthesis; HMF; immobilization; isopropylamine;
reuse stability
1. Introduction
Amines, particularly chiral amines, are crucial building blocks in the pharmaceuti-
cal industry, with approx. 40–45% of drug candidates containing an amine function [
1
].
Additionally, compounds with amine functionality also have applications in dyes, poly-
mers, and agrochemicals [
2
,
3
]. While chiral amines are still commonly produced chemi-
cally, e.g., by reductive amination of ketones (e.g., [
4
]), biocatalysis offers a more sustain-
able alternative for their production in line with the principles of Green Chemistry [
5
,
6
].
Amine transaminases (ATAs), next to amine oxidases [
7
,
8
], imine reductases [
9
,
10
], or
lipases [
11
,
12
], are attracting growing interest for large-scale synthesis of (chiral) amines
because they offer high enantioselectivity, internal cofactor regeneration, and can accept a
variety of different substrates. Amine transaminases transfer the amino-group of an amino
Catalysts 2023,13, 875. https://doi.org/10.3390/catal13050875 https://www.mdpi.com/journal/catalysts
Catalysts 2023,13, 875 2 of 15
donor to an amine acceptor using the cofactor pyridoxal-5
0
-phopsphate (PLP), regardless
of the position of the functional group, and unlike
α
-amino acid transaminases, they can
operate regardless of the presence of a carboxyl group [13–16].
The sustainability of the enzymatic process can be further increased by using re-
newable materials. As such, furfurals have gained increasing interest in recent years as
they can be produced from renewable raw materials and serve as interesting building
blocks for various chemicals as well as bio-based fuels [
17
–
25
]. As a result, furfurals are
among the top 30 value-added chemicals from biomass, according to the US Department
of Energy [
6
]. In particular, furfurylamines are relevant intermediates in the produc-
tion of pharmaceuticals, including diuretics [
26
] and antiseptics [
27
], or as intermediates
for fungicides [
28
], herbicides, and pesticides [
29
]. Two furfurylamines of interest are
5-(hydroxymethyl)furfurylamine (HMFA) and 2,5-di(aminomethyl)furan (DAF), which
are generated from 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF), re-
spectively. Both HMF and DFF can be produced from renewable resources. HMF occurs
naturally in products such as honey, toasted bread, or coffee [
20
,
30
–
32
], and it can be
synthesized from fructose, isomerized from glucose [
33
], or retrieved sustainably from food
waste biomass [
34
,
35
]. DFF, which can be used as a crosslinker for enzyme immobiliza-
tion [
36
], can be produced from fructose or glucose [
37
]. Both HMFA and DAF are relevant
building blocks in the pharmaceutical industry. Moreover, DAF, with its two terminal
amino groups, is an interesting compound for the polymer industry, as it can be used as a
raw material for polyamides [
3
], polyureas [
38
], and polyurethanes [
39
]. To date, only a
few publications have focused on the enzymatic amination of HMF and DFF [
17
,
24
,
40
,
41
],
while most studies aim at the chemical synthesis of HMFA and DAF (e.g., [
18
,
21
,
42
,
43
]).
For biocatalysis to become an attractive and competitive alternative to conventional chemi-
cal synthesis, a stable and easy-to-use enzyme is necessary [
44
]. Several research efforts
have been made to increase the stability of enzymes, for example by genetic mutation or
adapting the reaction environment [
45
–
50
]. One popular strategy is immobilization of
the enzymes, which has shown promising results in increasing the stability of different
transaminases under various conditions so far [
51
–
53
]. Moreover, immobilization of the
catalyst allows for easy removal from the reaction solution, reuse of the enzyme for several
reaction cycles or in enzyme cascades, and, in some cases, modification of the stability
and activity [
35
–
47
,
51
,
52
,
54
–
64
]. However, as each enzyme exhibits a different behavior
when immobilized, the immobilization process needs to be newly established for each
biocatalyst [
48
]. In general, immobilization can be achieved through a vast variety of
techniques and with various materials and is mainly divided into two groups: carrier-
dependent or carrier-free immobilized enzymes [
55
]. While carrier-free immobilization is
possible through crosslinking of the enzymes themselves, carrier-bound immobilization
requires a carrier such as a bead, fiber, capsule, film, or membrane [
55
]. The enzyme is either
adsorbed on the carrier or covalently bound to it using a crosslinker such as glutaralde-
hyde [
65
], bisepoxide [
66
], or DFF [
36
]. Many carrier materials have been successfully
applied for the immobilization of transaminases, including silica [
56
], chitosan [
60
,
67
],
resins [
51
,
61
,
66
], epoxy beads [
57
], cellulose [
68
], PVA-Fe3O4 nanoparticles [
69
], MnO
2
nanorods [70], controlled porosity glass [71,72], or lignin [73].
Another major benefit of using immobilized enzymes for biocatalysis is the possibility
of using the catalyst in a continuously operated flow reactor. Compared to batch catalysis,
catalysis in flow offers benefits such as reduced product inhibition, continuous product
removal, or improved heat and mass transfer [
74
,
75
]. Immobilized transaminases have
been used in continuous flow reactions for the kinetic resolution of amines [
56
,
66
,
72
,
76
]
or the synthesis of amines [
68
,
73
,
77
–
85
]. In this study, different ATAs were used for the
reductive amination of HMF and DFF to HMFA and DAF, respectively, using alanine and
isopropylamine as amine donors (Scheme 1). These ATAs have been immobilized using two
different strategies (i.e., site-selective and multiple binding to amine and glutaraldehyde-
functionalized amine beads, respectively) and analyzed in batch synthesis of HMFA and
DAF and in continuous-flow synthesis of HMFA on a preparative scale.
Catalysts 2023,13, 875 3 of 15
Catalysts2023,13,x 3of16
twodifferentstrategies(i.e.,site‐selectiveandmultiplebindingtoamineandglutaralde‐
hyde‐functionalizedaminebeads,respectively)andanalyzedinbatchsynthesisofHMFA
andDAFandincontinuous‐flowsynthesisofHMFAonapreparativescale.
Scheme1.Reactionschemeforthetransaminase‐catalyzedaminationof5‐(hydroxymethyl)furfural
(HMF)and2,5‐diformylfuran(DFF)usingisopropylamineoralanineasaminedonors,yielding5‐
(hydroxymethyl)furfurylamine(HMFA)or2,5‐di(aminomethyl)furan(DAF),respectively,andace‐
toneorpyruvateasco‐product.
2.ResultsandDiscussion
2.1.ActivityofSolubleATAstowardsHMFandDFF
Fourdifferentaminetransaminases(ATAs)werestudiedfortheiractivitytowards
HMFandDFFassubstrateswithL‐alanineasco‐substrate:Three(S)‐selectiveATAsfrom
Vibriofluvialis(ATA‐Vfl),Burkholderiamultivorans(ATA‐Bmu),andSilicibacterpomeroyi
(ATA‐Spo),andone(R)‐selectiveATAfromLuminiphilussyltensisNOR5‐1B(ATA‐Lsy)
(FigureS1).AstheATAsusedmayhavedifferentpHprofilesfortheaminationofHMF
andDFF,anappropriatepHforallreactionscatalyzedbytheATAsshouldbeevaluated
forcomparisonpurposes.Therefore,theconversionofbothsubstrateswasanalyzedat
differentpHvalues(pH6–11with1.0‐steps,FigureS2)withalanineasaco‐substrate.The
decreaseintheconcentrationofHMFandDFFwasfollowedspectrophotometrically.All
fourtransaminasesappearedtoexhibitthesameoptimumatpH8.0–9.0forbothsub‐
strates,withaverysimilarpHprofile.However,thespecificactivitiesofthesetransami‐
nasesdifferedsignificantly.WhileATA‐VflandATA‐Bmushowedthehighestactivities
forbothsubstrates,ATA‐SpoandATA‐Lsywerelessactive(Table1,FigureS2).Inaddi‐
tion,thespecificactivitiesweregenerallyhigherwhenHMFwasusedasasubstratecom‐
paredtoDFF,whichcouldbeattributedtothetwoaldehydegroupspresentinDFF,
whichrequiretwoaminationsteps.
Table1.SpecificactivityofsolubleATAswithHMFandDFFassubstrates.Thespecificactivityof
solubleATAs(ATA‐Vfl,ATA‐Bmu,ATA‐Spo,andATA‐Lsy)wasdeterminedatdifferentpHsas
describedinFigureS2andlistedhereforpH8.0(atwhichthestandardreactionisperformed)and
9.0(pH‐optimumformostoftheATAsused).Experimentswereperformedintriplicate.
TransaminaseSubstrateSpecificActivityatpH8.0
[mU/mgEnzyme]
SpecificActivityatpH9.0
[mU/mgEnzyme]
ATA‐VflHMF105.3±0.3126.1±0.5
DFF113.1±3.196.1±0.7
ATA‐BmuHMF117.0±1.0138.4±4.2
DFF63.3±1.769.1±0.3
ATA‐SpoHMF48.4±0.157.7±0.5
DFF39.1±0.847.8±1.5
ATA‐LsyHMF30.8±0.746.1±3.0
DFF27.3±0.434.8±2.4
Scheme 1.
Reaction scheme for the transaminase-catalyzed amination of 5-(hydroxymethyl)furfural
(HMF) and 2,5-diformylfuran (DFF) using isopropylamine or alanine as amine donors, yielding
5-(hydroxymethyl)furfurylamine (HMFA) or 2,5-di(aminomethyl)furan (DAF), respectively, and
acetone or pyruvate as co-product.
2. Results and Discussion
2.1. Activity of Soluble ATAs towards HMF and DFF
Four different amine transaminases (ATAs) were studied for their activity towards
HMF and DFF as substrates with L-alanine as co-substrate: Three (S)-selective ATAs from
Vibrio fluvialis (ATA-Vfl), Burkholderia multivorans (ATA-Bmu), and Silicibacter pomeroyi
(ATA-Spo), and one (R)-selective ATA from Luminiphilus syltensis NOR5-1B (ATA-Lsy)
(Figure S1). As the ATAs used may have different pH profiles for the amination of HMF
and DFF, an appropriate pH for all reactions catalyzed by the ATAs should be evaluated
for comparison purposes. Therefore, the conversion of both substrates was analyzed at
different pH values (pH 6–11 with 1.0-steps, Figure S2) with alanine as a co-substrate. The
decrease in the concentration of HMF and DFF was followed spectrophotometrically. All
four transaminases appeared to exhibit the same optimum at pH 8.0–9.0 for both substrates,
with a very similar pH profile. However, the specific activities of these transaminases
differed significantly. While ATA-Vfl and ATA-Bmu showed the highest activities for both
substrates, ATA-Spo and ATA-Lsy were less active (Table 1, Figure S2). In addition, the
specific activities were generally higher when HMF was used as a substrate compared to
DFF, which could be attributed to the two aldehyde groups present in DFF, which require
two amination steps.
Table 1.
Specific activity of soluble ATAs with HMF and DFF as substrates. The specific activity
of soluble ATAs (ATA-Vfl, ATA-Bmu, ATA-Spo, and ATA-Lsy) was determined at different pHs as
described in Figure S2 and listed here for pH 8.0 (at which the standard reaction is performed) and
9.0 (pH-optimum for most of the ATAs used). Experiments were performed in triplicate.
Transaminase Substrate Specific Activity at pH 8.0
[mU/mg Enzyme]
Specific Activity at pH 9.0
[mU/mg Enzyme]
ATA-Vfl HMF 105.3 ±0.3 126.1 ±0.5
DFF 113.1 ±3.1 96.1 ±0.7
ATA-Bmu HMF 117.0 ±1.0 138.4 ±4.2
DFF 63.3 ±1.7 69.1 ±0.3
ATA-Spo HMF 48.4 ±0.1 57.7 ±0.5
DFF 39.1 ±0.8 47.8 ±1.5
ATA-Lsy HMF 30.8 ±0.7 46.1 ±3.0
DFF 27.3 ±0.4 34.8 ±2.4
2.2. Immobilization of ATAs
Next, the immobilization of ATA-Spo on amine (HA)- and glutaraldehyde-func-
tionalized amine (HA
GA
) beads was analyzed to establish optimal immobilization condi-
tions, which has already been carried out previously for the other three ATAs [
57
]. For
HA-immobilization, ATAs with the aldehyde tag (encoding the amino acid sequence
Catalysts 2023,13, 875 4 of 15
CTPSR) [
86
] were incubated with the formylglycine-generating enzyme (FGE) to con-
vert the cysteine to the unique C
α
-formylglycine [
87
,
88
], which interacts with the amine
functions exposed on the beads to allow site-selective and targeted immobilization. In
contrast, non-tagged ATAs were immobilized on HA
GA
beads by multiple bindings of
exposed amino acid residues (especially lysines) [
89
,
90
] to the aldehyde functions exposed
on the beads. In general, ATA-Spo was optimally immobilized at 22
◦
C, at a pH of 5.0
(HAGA-) or 7.5 (HA and HAGA-immobilization), with 100 µg (HAGA-) or 150 µg (HA and
HA
GA
-immobilization) enzyme per mg bead, and with a short duration of 4 h (Figure S3).
These conditions were similar to those established for the other ATAs used in this study
when immobilized on HA- and HA
GA
beads [
57
]. Using the optimized immobilization
conditions for each ATA, specific immobilization parameters were evaluated in this study
(Table 2). The immobilization parameters (i.e., specific activity of the immobilizates, bind-
ing efficiency, and activity recovery) of ATA-Vfl, ATA-Bmu, and ATA-Lsy in this study were
comparable to those previously reported [
57
], and the parameters of newly immobilized
ATA-Spo immobilization were likewise similar. In general, with regard to the specific
activities of the immobilized enzymes, immobilization on HA
GA
beads (>50 U/mg
bead
)
appeared to be superior to immobilization on HA beads for all (S)-selective ATAs stud-
ied here. In contrast, the (R)-selective ATA-Lsy exhibited higher specific activity when
immobilized on HA beads (52 U/mgbead) compared with HAGA beads (34 U/mgbead).
Table 2.
Immobilization parameters. To evaluate various immobilization parameters, each transami-
nase was immobilized on a larger scale (280 mg beads) using the optimized conditions (Table 1), and
protein concentration and activity were determined in all solutions. The specific activities of soluble
and immobilized ATAs were determined as described in the methods using rac-1-PEA as substrate
(50 mM Tris pH 8.0, 2.5 mM rac-1-PEA, 2.5 mM pyruvate, 0.1 mM PLP, 0.5% DMSO) at 37 ◦C.
Transaminase
Specific Activity of
Soluble Enzyme [a]
[U/mg Enzyme]
Bead Type
Specific Activity of
Immobilized Enzyme [b]
[U/g Bead]
Binding
Efficiency [c]
[%]
Activity
Recovery [d]
[%]
ATA-Vfl 4.2 HA 48.6 74.5 10.6
HAGA 56.3 61.9 9.7
ATA-Bmu 2.0 HA 26.6 97.6 18.2
HAGA 51.8 71.1 17.7
ATA-Spo 0.8 HA 35.8 92.5 14.2
HAGA 57.6 93.8 12.4
ATA-Lsy 1.0 HA 52.3 75.3 14.4
HAGA 33.6 96.2 16.1
[a]
The specific activities of soluble ATA-Vfl, ATA-Bmu, and ATA-Lsy were previously determined under equal
conditions [
57
]. The specific activities of the soluble non-tagged ATAs (used for HA
GA
bead immobilization)
did not change by adding the aldehyde tag (used for HA bead immobilization), and their reaction behavior was
similar; hence, only one activity is listed.
[b]
The specific activity of the immobilized enzyme is the observed
activity of the immobilized ATA per g of bead support.
[c]
The binding efficiency is the percentage ratio between
the total amount of immobilized enzyme (the protein amount in the starting solution minus the protein amount in
the supernatant) and the total amount of protein initially applied for immobilization.
[d]
The activity recovery
is the percentage ratio between the observed total activity of the immobilized biocatalyst and the total activity
initially applied for immobilization.
2.3. Amination of HMF and DFF Using Immobilized ATA-Spo in Batch
To analyze the amination of HMF and DFF by an immobilized transaminase, ATA-Spo
immobilized on HA
GA
beads was selected to be used in batch synthesis with L-alanine or
isopropylamine as a co-substrate (Figure 1). In general, HMF conversion was much faster
than DFF conversion for both co-substrates, which is consistent with the lower activity
of soluble ATAs towards DFF. However, it must be considered that DFF undergoes two
amination steps via an aminoaldehyde intermediate. Interestingly, the initial reaction rate
for HMF and DFF was lower with isopropylamine compared to alanine, which may be
attributed to better acceptance of alanine by transaminases, as is often observed [
91
,
92
].
Catalysts 2023,13, 875 5 of 15
However, with increasing reaction time, the conversion of both substrates became more effi-
cient with isopropylamine (i.e., the conversion was faster), and furthermore, both substrates
were aminated to a higher extent (88% (HMFA) and 88% (DAF) vs. >99.5% (HMFA) and
99% (DAF) conversion of HMF to HMFA and DFF to DAF with alanine and isopropylamine
as co-substrate, respectively). Other reports of transaminases applied in soluble form for
these reactions show comparable or lower conversion. Dunbabin et al., reached up to ap-
prox. 89% (HMFA) and 70% (DAF) conversion after 24 h using 1-phenylethylamine (5-fold
excess) and isopropylamine (10-fold excess) as co-substrate [
17
]. Interestingly, in this study,
1-phenylethylamine was a better co-substrate for DFF conversion, whereas isopropylamine
was better for the conversion of HMF in most other cases. Wang et al. achieved 93.2%
HMFA with L-alanine (15-fold excess) [
41
] and Gao et al. reached a comparable conversion
of 97.7% HMFA with D-alanine (24-fold excess) [40], both using whole cells.
Catalysts2023,13,x 5of16
2.3.AminationofHMFandDFFUsingImmobilizedATA‐SpoinBatch
ToanalyzetheaminationofHMFandDFFbyanimmobilizedtransaminase,ATA‐
SpoimmobilizedonHAGAbeadswasselectedtobeusedinbatchsynthesiswithL‐alanine
orisopropylamineasaco‐substrate(Figure1).Ingeneral,HMFconversionwasmuch
fasterthanDFFconversionforbothco‐substrates,whichisconsistentwiththelowerac‐
tivityofsolubleATAstowardsDFF.However,itmustbeconsideredthatDFFundergoes
twoaminationstepsviaanaminoaldehydeintermediate.Interestingly,theinitialreaction
rateforHMFandDFFwaslowerwithisopropylaminecomparedtoalanine,whichmay
beattributedtobetteracceptanceofalaninebytransaminases,asisoftenobserved[91,92].
However,withincreasingreactiontime,theconversionofbothsubstratesbecamemore
efficientwithisopropylamine(i.e.,theconversionwasfaster),andfurthermore,bothsub‐
strateswereaminatedtoahigherextent(88%(HMFA)and88%(DAF)vs.>99.5%(HMFA)
and99%(DAF)conversionofHMFtoHMFAandDFFtoDAFwithalanineandisoprop‐
ylamineasco‐substrate,respectively).Otherreportsoftransaminasesappliedinsoluble
formforthesereactionsshowcomparableorlowerconversion.Dunbabinetal.,reached
uptoapprox.89%(HMFA)and70%(DAF)conversionafter24husing1‐phenylethyla‐
mine(5‐foldexcess)andisopropylamine(10‐foldexcess)asco‐substrate[17].Interest‐
ingly,inthisstudy,1‐phenylethylaminewasabetterco‐substrateforDFFconversion,
whereasisopropylaminewasbetterfortheconversionofHMFinmostothercases.Wang
etal.achieved93.2%HMFAwithL‐alanine(15‐foldexcess)[41]andGaoetal.reacheda
comparableconversionof97.7%HMFAwithD‐alanine(24‐foldexcess)[40],bothusing
wholecells.
Thelowerconversioninreactionswithalanineasco‐substratecouldbeduetoprod‐
uctinhibitionbypyruvate,whichhasbeenobservedwithvarioustransaminases[93,94],
andtoanunfavorableequilibriumonthesubstrateside,whichisoftenobserved,forex‐
ample,intransaminase‐catalyzedasymmetricsynthesisreactions[95,96].Productinhibi‐
tionandunfavorableequilibriummaybecounteractedbyusingisopropylamineasaco‐
substrateandremovingtheco‐product,acetone,throughevaporation,whichhasalready
beensuccessfullyaddressedinotherstudies[97,98].
Figure1.AminationofHMFandDFFbyimmobilizedATA‐Spoinbatchreaction.ATA‐Spowas
immobilizedonHAGAbeads(5mgeach)andusedfortheaminationofHMFandDFFin1mL
reactionsolution(50mMHEPESpH8.0,13mMHMForDFF,500mMisopropylamineorL‐alanine,
0 100 200 300 400 500
0
20
40
60
80
100
conversion [%]
time [min]
HMF + L-alanine DFF + L-alanine
HMF + isopropylamine DFF + isopropylamine
Figure 1.
Amination of HMF and DFF by immobilized ATA-Spo in batch reaction. ATA-Spo was
immobilized on HA
GA
beads (5 mg each) and used for the amination of HMF and DFF in 1 mL
reaction solution (50 mM HEPES pH 8.0, 13 mM HMF or DFF, 500 mM isopropylamine or L-alanine,
0.1 mM PLP) in 2 mL reaction vessels at 37
◦
C with shaking. The reaction was followed by the
detection of HMF and DFF in diluted supernatant at 283 nm, and the initial concentration was set at
100%. All reactions were performed as duplicates, and the error bars represent the maximum and
minimum. (
: HMF and L-alanine;
•
: HMF and isopropylamine;
N
: DFF; and L-alanine
H
: DFF
and isopropylamine).
The lower conversion in reactions with alanine as co-substrate could be due to product
inhibition by pyruvate, which has been observed with various transaminases [
93
,
94
], and
to an unfavorable equilibrium on the substrate side, which is often observed, for example,
in transaminase-catalyzed asymmetric synthesis reactions [
95
,
96
]. Product inhibition and
unfavorable equilibrium may be counteracted by using isopropylamine as a co-substrate
and removing the co-product, acetone, through evaporation, which has already been
successfully addressed in other studies [97,98].
2.4. Reusability of Immobilized ATAs for Amination of HMF Using Alanine or Isopropylamine
Immobilization of enzymes offers the major advantage of reusability of the catalysts
for multiple reaction cycles. Moreover, in a continuous flow reactor, enzyme immobilization
is necessary, and high reusability stability is desirable [
99
,
100
]. To analyze the reusability
Catalysts 2023,13, 875 6 of 15
potential of the immobilized ATAs, repeated cycles of HMF amination with an excess of
isopropylamine or alanine as co-substrate were performed for 4 h each at 37 ◦C.
When alanine was used as the amine donor (Figure 2a), high conversion (>99%) as
well as high reusability stability (little to no reduction in conversion) were observed in
all cases, except for ATA-Bmu immobilized on HA beads and ATA-Lsy immobilized on
HA
GA
beads. Thus, alanine as a co-substrate seems to be a good choice as an amine donor,
as it leads to high conversion and high reuse stability for most of the ATAs studied here.
Furthermore, alanine is accepted by most transaminases and is generally considered an
environmentally friendly and sustainable chemical. The high reuse stability of ATA-Vfl,
ATA-Bmu, and ATA-Lsy was also observed when 1-phenylethylamine (1-PEA) was used as
a substrate and alanine as a co-substrate [
57
]. In contrast, isopropylamine (Figure 2b) had a
significant negative effect on the reusability of most immobilized ATAs. The conversion
of HMF to HMFA catalyzed by ATA-Vfl gradually decreased with each cycle, dropping
from 98% and 90% in the first cycle to 36% and 35% in the fifth cycle for HA
GA
- and
HA-immobilizates, respectively. In the case of ATA-Bmu immobilized on HA
GA
beads,
the conversion decreased from 83% in the first cycle to 63% in the fifth cycle, indicating
the destabilizing effects of isopropylamine, but was lower compared to ATA-Vfl. The
latter may be due to the generally high operational and solvent stability described for the
tetrameric ATA-Bmu [
101
]. However, when immobilized on HA beads, the conversion was
very low (13%) from the first cycle, leading to the assumption that ATA-Bmu is destabilized
either by the C-terminally added aldehyde tag or by the C-terminally oriented site-selective
immobilization. ATA-Lsy does not appear to accept isopropylamine as an amine donor
substrate, as no conversion was observed with either immobilizate. They showed high
activity when alanine was used. Interestingly, ATA-Spo immobilized on both HA
GA
and
HA beads yielded the highest conversion of HMF to HMFA using isopropylamine as a
co-substrate, with no reduction in conversion observed over all five cycles. Thus, ATA-Spo
appeared to accept isopropylamine as a co-substrate best and to have the highest reuse
stability compared with the other ATAs studied. In general, various transaminases do
not accept isopropylamine at all or accept it poorly, resulting in low conversion of the
ketone substrate (here HMF) [
102
–
104
]. Consistent with the results shown here, ATA-
Spo and ATA-Bmu have shown tolerance to isopropylamine in other studies at higher
concentrations as co-substrate for the amination of various substrates, resulting in high
conversion of the substrates [
92
,
105
] In contrast, ATA-Vfl has been shown not to accept
isopropylamine well [
106
], which explains the rapid decrease in the conversion of HMF
with isopropylamine as a co-substrate with each cycle.
Since ATA-Spo immobilized on HA
GA
beads exhibited the highest stability with
respect to its reuse and the use of isopropylamine as a co-substrate, it was selected for
further analysis in continuous-flow catalysis despite its lower activity compared to the
other ATAs studied.
2.5. Amination of HMF in Continuous Flow Using Immobilized ATA-Spo with Alanine
or Isopropylamine
Finally, the performance of ATA-Spo immobilized on HA
GA
beads in a continuous-
flow reactor was investigated. For this purpose, the reaction solution containing 10 mM
HMF was passed through the reactor containing the immobilizates (50 mg) at 21
◦
C with a
flow rate of 50
µ
L/min to achieve a residence time of approx. 4 min in a reactor volume of
236
µ
L (Figure 3). The column and feed solution were kept dark to avoid the inactivating
effect of light on ATA-Spo [
49
,
50
]. The protein concentration in the flow-through was
analyzed with the Bradford Assay, but no detectable enzyme leaching was observed.
The residence time of 4 min was sufficient to achieve a high initial conversion of HMF
to HMFA in the first 4 and 24 h when either L-alanine (98% and 95%, respectively) or
isopropylamine (75% and 67%, respectively) were used as amine donors, with significantly
higher conversion after up to 4 days when L-alanine was used. The latter observation can
be attributed to the higher reaction velocity observed before when using L-alanine in batch
Catalysts 2023,13, 875 7 of 15
synthesis as a co-substrate (Figure 1), leading to a faster conversion in the continuous-
flow mode and thus a higher total production of HMFA. To investigate the stability of
the immobilized ATA, the flow reactor was run for 12 days, and samples were analyzed
every day, showing that the conversion of HMF continuously decreased over time in both
cases, reaching 48% and 41% after 12 days when using L-alanine and isopropylamine as co-
substrate, respectively. The gradual decrease over time is likely caused by the inactivation
of the immobilized enzyme, whose operational stability under the given conditions was
not high enough. High amine concentrations, as present in the feed solution, tend to
destabilize transaminases [
44
,
107
]. The initial conversion was comparable to other studies
using alanine and isopropylamine as co-substrate in reductive aminations catalyzed by
immobilized transaminase [
80
,
85
,
108
]. Importantly, in other studies, the short- and long-
term conversion was optimized by a higher temperature, a longer residence time, and/or a
higher amount of immobilizate per reactor volume, which may also increase the conversion
of HMF in this study. Using the initial conversion (day 1) of the continuous-flow synthesis
of HMFA, a space-time yield of 8 mg
×
mL
−1×
h
1
was achieved when using either L-
alanine or isopropylamine as co-substrate and a half-life of 11 days (using L-alanine) and
9.5 days (using isopropylamine).
Catalysts2023,13,x 7of16
Figure2.ReusabilityofATA‐immobilizatesforHMFamination.Thetransaminases(ATA‐Vfl,ATA‐
Bmu,ATA‐Spo,andATA‐Lsy)wereimmobilizedseparatelyonHA‐orHAGAbeads(5mgeach)
andusedfortheaminationofHMFtoHMFAinfiverepetitivecycleswithalanine(a)orisopropyl‐
amine(b)astheco‐substrate.Aftereachcycle,thesupernatantwasremoved,andtheconversionof
HMFwasdeterminedusinganalyticalHPLCasdescribedinthemethods.Subsequently,thebeads
werewashedthreetimeswithwaterandincubatedwithanewreactionsolution(1mLof50mM
HEPESpH8.0,10mMHMF,500mMisopropylamineoralanine(L‐orD‐alaninefor(S)‐or(R)‐selec‐
tiveATAs,respectively),0.1mMPLP)for4hat37°Cwithshaking.Allreactionswereperformed
asduplicates,andtheerrorbarsrepresentthemaximumandminimum.
2.5.AminationofHMFinContinuousFlowUsingImmobilizedATA‐SpowithAlanineor
Isopropylamine
Finally,theperformanceofATA‐SpoimmobilizedonHAGAbeadsinacontinuous‐
flowreactorwasinvestigated.Forthispurpose,thereactionsolutioncontaining10mM
HMFwaspassedthroughthereactorcontainingtheimmobilizates(50mg)at21°Cwith
aflowrateof50μL/mintoachievearesidencetimeofapprox.4mininareactorvolume
of236μL(Figure3).Thecolumnandfeedsolutionwerekeptdarktoavoidtheinactivat‐
ingeffectoflightonATA‐Spo[49,50].Theproteinconcentrationintheflow‐throughwas
analyzedwiththeBradfordAssay,butnodetectableenzymeleachingwasobserved.The
residencetimeof4minwassufficienttoachieveahighinitialconversionofHMFto
HMFAinthefirst4and24hwheneitherL‐alanine(98%and95%,respectively)oriso‐
propylamine(75%and67%,respectively)wereusedasaminedonors,withsignificantly
higherconversionafterupto4dayswhenL‐alaninewasused.Thelatterobservationcan
ATA-Vfl
HA
ATA-Vfl
HA
GA
ATA-Bmu
HA
ATA-Bmu
HA
GA
ATA-Spo
HA
ATA-Spo
HA
GA
ATA-Lsy
HA
ATA-Lsy
HA
GA
0
20
40
60
80
100
conversion [%]
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5
Co-Substrate: Alanine
ATA-Vfl
HA
ATA-Vfl
HA
GA
ATA-Bmu
HA
ATA-Bmu
HA
GA
ATA-Spo
HA
ATA-Spo
HA
GA
ATA-Lsy
HA
ATA-Lsy
HA
GA
0
20
40
60
80
100
conversion [%]
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5
Co-Substrate: Isopropylamine
(a)
(b)
Figure 2. Reusability of ATA-immobilizates for HMF amination. The transaminases (ATA-Vfl, ATA-
Bmu, ATA-Spo, and ATA-Lsy) were immobilized separately on HA- or HA
GA
beads (5 mg each) and
used for the amination of HMF to HMFA in five repetitive cycles with alanine (
a
) or isopropylamine
(
b
) as the co-substrate. After each cycle, the supernatant was removed, and the conversion of HMF
was determined using analytical HPLC as described in the methods. Subsequently, the beads were
washed three times with water and incubated with a new reaction solution (1 mL of 50 mM HEPES
pH 8.0, 10 mM HMF, 500 mM isopropylamine or alanine (L- or D-alanine for (S)- or (R)-selective
ATAs, respectively), 0.1 mM PLP) for 4 h at 37
◦
C with shaking. All reactions were performed as
duplicates, and the error bars represent the maximum and minimum.
Catalysts 2023,13, 875 8 of 15
Catalysts2023,13,x 8of16
beattributedtothehigherreactionvelocityobservedbeforewhenusingL‐alanineinbatch
synthesisasaco‐substrate(Figure1),leadingtoafasterconversioninthecontinuous‐flow
modeandthusahighertotalproductionofHMFA.Toinvestigatethestabilityoftheim‐
mobilizedATA,theflowreactorwasrunfor12days,andsampleswereanalyzedevery
day,showingthattheconversionofHMFcontinuouslydecreasedovertimeinbothcases,
reaching48%and41%after12dayswhenusingL‐alanineandisopropylamineasco‐sub‐
strate,respectively.Thegradualdecreaseovertimeislikelycausedbytheinactivationof
theimmobilizedenzyme,whoseoperationalstabilityunderthegivenconditionswasnot
highenough.Highamineconcentrations,aspresentinthefeedsolution,tendtodestabi‐
lizetransaminases[44,107].Theinitialconversionwascomparabletootherstudiesusing
alanineandisopropylamineasco‐substrateinreductiveaminationscatalyzedbyimmo‐
bilizedtransaminase[80,85,108].Importantly,inotherstudies,theshort‐andlong‐term
conversionwasoptimizedbyahighertemperature,alongerresidencetime,and/ora
higheramountofimmobilizateperreactorvolume,whichmayalsoincreasetheconver‐
sionofHMFinthisstudy.Usingtheinitialconversion(day1)ofthecontinuous‐flow
synthesisofHMFA,aspace‐timeyieldof8mg×mL−1×h1wasachievedwhenusingeither
L‐alanineorisopropylamineasco‐substrateandahalf‐lifeof11days(usingL‐alanine)and
9.5days(usingisopropylamine).
Figure3.AminationofHMFbyimmobilizedATA‐Spoinacontinuous‐flowreactor.ATA‐Spo,im‐
mobilizedon50mgHAGAbeads,wasusedfortheaminationof10mMHMFtoHMFAinacontin‐
uousflowwithisopropylamine(●)orL‐alanine(■)asco‐substrate.Thereactionsolution(50mM
HEPESpH8.0,10mMHMF,500mMco‐substrate,0.1mMPLP)wasappliedataconstantflowrate
of50μL/min.Theresidencetimeofthereactionsolutioninthereactorcolumnwasapprox.4min.
Thereactionwasperformedat21°C,andtheconversionwasdeterminedbyquantificationofHMF
andHMFAintheflowthroughusinganalyticalHPLCasdescribedintheMaterialsandMethods.
3.MaterialsandMethods
3.1.GeneralInformation
Allchemicalsandsolventsusedwereobtainedmainlyfromcommercialdistributors
ofanalyticalgrade:Merck(Darmstadt,Germany),VWR(Radnor,PA,USA),CarlRoth
(Karlsruhe,Germany),andThermoFisherScientific(Waltham,MA,USA).Theamine
(ReliZymeTM,HA403,abbreviatedasHA‐)beadswerepurchasedinM‐sizefromResin‐
024681012
0
20
40
60
80
100
conversion [%]
time [d]
L-Alanine
Isopropylamine
Figure 3.
Amination of HMF by immobilized ATA-Spo in a continuous-flow reactor. ATA-Spo,
immobilized on 50 mg HA
GA
beads, was used for the amination of 10 mM HMF to HMFA in a
continuous flow with isopropylamine (
•
) or L-alanine (
) as co-substrate. The reaction solution
(50 mM HEPES pH 8.0, 10 mM HMF, 500 mM co-substrate, 0.1 mM PLP) was applied at a constant
flow rate of 50
µ
L/min. The residence time of the reaction solution in the reactor column was approx.
4 min. The reaction was performed at 21
◦
C, and the conversion was determined by quantifica-
tion of HMF and HMFA in the flowthrough using analytical HPLC as described in the Materials
and Methods.
3. Materials and Methods
3.1. General Information
All chemicals and solvents used were obtained mainly from commercial distributors
of analytical grade: Merck (Darmstadt, Germany), VWR (Radnor, PA, USA), Carl Roth
(Karlsruhe, Germany), and Thermo Fisher Scientific (Waltham, MA, USA). The amine
(ReliZyme
TM
, HA 403, abbreviated as HA-) beads were purchased in M-size from Resindion
S.r.l. (Binasco, Italy) 2,5-diformylfuran (DFF) was chemically synthesized by oxidation of
fructose following the literature protocol [
37
]. The product was obtained with 99% purity,
as determined by 1H-NMR in CDCl3.
3.2. Preparation of Glutaraldehyde-Functionalized Amine Beads
The amine beads were functionalized with glutaraldehyde (abbreviated as HA
GA
beads) exactly as described previously [57].
3.3. Cloning of the Aldehyde-tagged ATA-Spo
Aldehyde-tagged ATAs (after FGly generation; see below) were used exclusively for
immobilization on the HA beads. The addition of the aldehyde tag to ATA-Vfl, ATA-
Bmu, and ATA-Lsy has been described previously [
57
,
86
], whereas the generation of the
aldehyde-tagged ATA-Spo was performed in this study according to the protocols used
for the other ATAs. Briefly, the coding sequence of ATA-Spo was amplified with add-on
primers (forward:
aaggagatatacatatg
agcctggcgaccattacg, reverse:
gagtacataaactagcact
ta
cttcagatttcatcagaccctg; underlined sequences: complementary sequences for the subsequent
overlap extinction (OE) PCR); italic sequences: sequences for the amplification of the coding
sequence of ATA-Spo) by add-on PCR (analogous to that described previously with an
annealing temperature in the first 4 cycles of 63
◦
C). Subsequently, the amplicons were used
as megaprimers in OE-PCR (exactly as described previously) to insert the sequence into the
Catalysts 2023,13, 875 9 of 15
previously generated plasmid pET24b-X-CTPSR-H6 (at position X with the C-terminally
aldehyde tag (CTPSR) and the His6tag (H6) [86].
3.4. Expression and Purification of Enzymes
The expression of
∆
72-hFGE was performed as described by Peng et al. [
87
]. The
purification of
∆
72-hFGE and the expression of the tagged and non-tagged ATA-Vfl, ATA-
Bmu, and ATA-Lsy were exactly performed as described previously [
57
]. Expression and
purification of the tagged and non-tagged ATA-Spo were performed analogously as for the
other ATAs.
3.5. Conversion of the Aldehyde Tag
The conversion of cysteine to C
α
-formylglycine within the aldehyde tag was catalyzed
by
∆
72-hFGE. Therefore,
∆
72-hFGE was added to the reaction solution (50 mM Tris pH 8.0,
0.1 mM PLP, 3 mM DTT) containing the tagged ATA in a molar ratio of 1:30 (
∆
72-hFGE:ATA)
and incubated for 4 h at 37
◦
C. Conversion was verified by fluorescent labeling with
Alexa Fluor
TM
488 Hydroxylamine, carried out exactly as previously described without
further purification [57].
3.6. Immobilization of ATAs on the Solid Supports
The establishment of the immobilization conditions and the final immobilization of
the ATAs on HA- and HAGA beads were exactly performed as described previously [57].
3.7. Activity Assay of Soluble and Immobilized ATAs
The specific activity of the ATAs was determined differently for each substrate. In the
case of 1-PEA, the product, acetophenone, was detected at 245 nm and quantified using the
extinction coefficient of 12 mM
−1
cm
−1
[
109
]. When HMF or DFF were used as substrates,
the decrease in absorbance of the substrates was detected at 283 nm and quantified using
the extinction coefficients of 14.922 mM
−1
cm
−1
(HMF) and 10.610 mM
−1
cm
−1
(DFF)
(Figure S4). Using soluble enzymes, the reactions were performed in UV-microtiter plates
(UV-Star
®
, Greiner Bio-One, Kremsmünster, Austria) containing the stated amounts of
ATA and a reaction volume of 150
µ
L (50 mM Tris pH 8.0, 0.5% DMSO, 0.1 mM PLP) with
the respective substrates (rac-1-PEA with pyruvate, HMF or DFF with isopropylamine
or alanine (L- or D-alanine for (S)- or (R)-selective ATAs, respectively) at the respective
indicated concentrations, and followed at the respective wavelength in the TECAN reader
(Spark
TM
10M, Männedorf, Switzerland) at 37
◦
C. In the case of the pH-screen, different
buffers were used instead of the one standard buffer mentioned above: 50 mM phosphate
buffer pH 6.0, 50 mM Tris buffer pH 7.0–9.0, or 50 mM CAPS buffer pH 10.0–11.0. Specific
activity was always determined at the initial reaction rate by using the average of 1 min
intervals in the first few minutes (generally 5–10 min). When using the immobilized
enzymes, 5 mg (the standard amount of beads unless stated otherwise) of the immobilizates
were shaken with 1.5 mL (the establishment of the immobilization) or 1.0 mL of reaction
solution (as indicated in each case) at 37
◦
C for 2 min. Subsequently, the analytes in the
diluted supernatant were determined at the respective wavelengths in the TECAN reader.
In the case of batch synthesis (Figure 1), the reaction was followed for 480 min, while the
conversion was followed by detecting the products as mentioned. The reactions catalyzed
by immobilized ATAs were performed in sealed 2 mL reaction tubes to avoid evaporation
of the products (i.e., acetophenone) [
110
]. One unit was defined as the amount of soluble or
immobilized enzyme that produced 1
µ
Mof the product in one minute at the mentioned
temperatures. All reactions were performed in duplicate and always started by adding the
reaction solution.
3.8. Batch Reusability Study
To investigate the reuse potential of each immobilized ATA in reductive amination of
HMF, 5 mg of each immobilizate were incubated in 1 mL reaction buffer (50 mM HEPES
Catalysts 2023,13, 875 10 of 15
buffer pH 8.0, 10 mM HMF, 0.1 mMPLP, and 500 mM amine donor (isopropylamine or L-
or D-alanine for (S)- or (R)-selective ATAs, respectively) at 37
◦
C for 4 h in 1 mL reaction
tubes under vigorous shaking. After each cycle, the reaction solution was removed, the
beads were washed with water, and they were used for another cycle. All experiments
were performed in duplicate and repeated for a total of five cycles. The concentration of
HMF and HMFA was analyzed using HPLC.
3.9. Continuous Flow Catalysis
ATA-Spo immobilized on 50 mg HA
GA
beads was added to a glass tube (inner diameter
10 mm, total length 100 mm, approx. height of bead layer 3 mm, approx. bead volume
0.235 mL), which was then filled with feed solution and attached to the pump (Metrohm
665 Dosimat, Herisau, Switzerland). The feed solution (50 mMHEPES buffer pH 8.0,
10 mM HMF, 0.1 mM PLP, and 500 mM amine donor (isopropylamine or L-alanine)) was
pumped through the tube with a flow rate of 50
µ
L min
−1
. The tube and the feed solution
were wrapped in aluminum foil to exclude negative influences from light [
49
,
50
]. Samples
of the flow were taken every 24 h and analyzed using HPLC.
3.10. HPLC Analysis
To quantify the concentration of the substrates and products, analytical HPLC analyses
were performed using a Waters 2695 Separations Module (Milford, MA, USA) with a Waters
2996 Photodiode Array Detector and a Supelco Discovery
®
(St. Louis, MO, USA) C18 column
(15 cm
×
4.6 mm). Therefore, 1 mL samples of batch and continuous flow catalysis experi-
ments were analyzed using an isocratic method (
98% acetonitrile + 2% ddH2O + 0.1% trifluo-
roacetic acid) at a constant flow of 1 mL min
−1
. Peaks were detected using a DAD detector
at 210 nm. Chromatograms in Figure S5.
3.11. Determination of Protein Concentration
For the determination of the total protein concentration in protein solutions, the Protein
Assay Dye Reagent Concentrate from BioRad (Hercules, CA, USA) (Art.5000006) was used
with bovine serum albumin as the calibration standard.
4. Conclusions
In this study, the biocatalytic reductive amination of HMF and DFF using amine
transaminases (ATAs) was performed. Four ATAs that exhibited activity towards HMF
and DFF were identified. Different pH values were tested for the ATAs, revealing that
slightly basic conditions led to the highest specific activity. Following, all four ATAs
were successfully immobilized on solid supports, with the (S)-selective ATAs (ATA-Vfl,
ATA-Bmu, and ATA-Spo) showing the highest specific activity when immobilized on
glutaraldehyde-functionalized amine (HA
GA
) beads via multi-attachment binding (56, 52,
and 58 U/g
bead
, respectively). On the other hand, immobilization on amine beads via
site-selective, oriented binding yielded the highest specific activity of the (R)-selective
ATA (ATA-Lsy, 52 U/g
bead
). Almost all of the immobilized ATAs demonstrated efficient
reusability when alanine was used as the amine donor for the reductive amination of
HMF. However, when isopropylamine was used, only ATA-Spo immobilized on both
supports and ATA-Bmu immobilized on HA
GA
beads revealed high reuse potential. High
conversion rates were achieved using ATA-Spo immobilized on HA
GA
beads in the batch
synthesis of HMFA and DAF with alanine as an amine donor (87% and 87%, respec-
tively) and almost complete conversion with isopropylamine as an amine donor (>99 %
and >98 %, respectively). Finally, the reductive amination of HMF in continuous-flow
mode using ATA-Spo immobilized on HA
GA
beads was conducted. High conversion was
achieved even after 12 days (48% and 41% when alanine and isopropylamine were used as
amine donors), with a half-life of 11 days with L-alanine and 9.5 days with isopropylamine.
Catalysts 2023,13, 875 11 of 15
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/catal13050875/s1, Figure S1: Amination of HMF by soluble ATAs;
Figure S2: Relative activity of different ATAs at different pH with HMF and DFF as substrate;
Figure S3: Analysis of various immobilization conditions for ATA-Spo; Figure S4: Characterization of
HMF, HMFA, DFF, and DAF., Figure S5: HPLC Analysis of HMFA and HMF.
Author Contributions:
Conceptualization, T.H., L.M.M., L.M.v.L. and P.B.; methodology, T.H.,
L.M.M. and J.L.; validation, T.H. and L.M.M.; formal analysis, T.H. and L.M.M.; investigation,
T.H., L.M.M. and J.L.; data curation, T.H. and L.M.M.; writing—original draft preparation, T.H.
and L.M.M.; writing—review and editing, all authors; visualization, T.H. and L.M.M.; supervision,
G.F.v.M., L.M.v.L., M.H., U.T.B. and P.B. All authors have read and agreed to the published version of
the manuscript.
Funding:
This project has received funding from the European Union’s Horizon 2020 research and
innovation program under the Marie Skłodowska-Curie grant agreement No. 860414.
Data Availability Statement: Data is contained within the article or Supplementary Materials.
Acknowledgments:
The authors would like to acknowledge Chiara Danielli (ViaZym B.V.) for syn-
thesizing DFF and her expertise on enzyme immobilization; Nils Berelsmann (University of Bielefeld)
for his help with the HPLC and UV/VIS measurements; and Kerstin Glüsenkamp (University of
Bielefeld) for her technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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