High performing immobilized Baeyer-Villiger monooxygenase and glucose dehydrogenase for the synthesis of ε-caprolactone derivative

Article abstract of DOI:10.1016/j.apcata.2018.12.036

The industrial application of Baeyer-Villiger monooxygenases (BVMOs) is typically hindered by stability and cofactor regeneration considerations. The stability of biocatalysts can be improved by immobilization. The goal of this study was to evaluate the (co)-immobilization of a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) with a glucose dehydrogenase (GDH) from Thermoplasma acidophilum for NADPH cofactor regeneration. Both enzymes were immobilized on an amino-functionalized agarose-based support (MANA-agarose). They were applied to the oxidation of 3,3,5-trimethylcyclohexanone for the synthesis of ε-caprolactone derivatives which are precursors of polyesters. The performances of the immobilized biocatalysts were evaluated in reutilization reactions with as many as 15 cycles and compared to the corresponding soluble enzymes. Co-immobilization proved to provide the most efficient biocatalyst with an average conversion of 83% over 15 reutilization cycles leading to a 50-fold increase of the biocatalyst yield compared to the use of soluble enzymes which were applied in a fed-batch strategy. TmCHMO was immobilized for the first time in this work, with very good retention of the activity throughout reutilization cycles. This immobilized biocatalyst contributes to the application of BVMOs in up-scaled biooxidation processes.

Full text of DOI:10.1016/j.apcata.2018.12.036

Accepted Manuscript
Title: High performing immobilized Baeyer-Villiger
monooxygenase and glucose dehydrogenase for the synthesis
of null-caprolactone derivative
Authors: Marie A.F. Delgove, Daniela Valencia, Jordi Sol e´ ,
Katrien V. Bernaerts, Stefaan M.A. De Wildeman, Marina
´
Guill e´ n, Gregorio Alvaro
PII:
S0926-860X(18)30632-X
DOI:
Reference:
https://doi.org/10.1016/j.apcata.2018.12.036
APCATA 16940
To appear in:
Applied Catalysis A: General
Received date:
Revised date:
Accepted date:
5 October 2018
10 December 2018
30 December 2018
Please cite this article as: Delgove MAF, Valencia D, Sol e´ J, Bernaerts KV,
´
De Wildeman SMA, Guill e´ n M, Alvaro G, High performing immobilized
Baeyer-Villiger monooxygenase and glucose dehydrogenase for the synthesis
of x190;-caprolactone derivative, Applied Catalysis A, General (2018),
https://doi.org/10.1016/j.apcata.2018.12.036
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High performing immobilized Baeyer-Villiger monooxygenase and glucose
dehydrogenase for the synthesis of Ɛ-caprolactone derivative
a
b
b
a
Marie A. F. Delgove , Daniela Valencia , Jordi Solé , Katrien V. Bernaerts , Stefaan M.
a
b
b
A. De Wildeman , Marina Guillén , Gregorio Álvaro
a
Maastricht University, Aachen-Maastricht Institute for Biobased Materials (AMIBM),
Brightlands Chemelot campus, Urmonderbaan 22, 6167 RD Geleen, The Netherlands
b
Bioprocess Engineering and Applied Biocatalysis Group, Department of Chemical,
Biological and Environmental Engineering, Universitat Autònoma de Barcelona, 08193
Bellaterra, Catalonia, Spain
Corresponding author:
Gregorio Álvaro
Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de
Barcelona, 08193 Bellaterra, Catalonia, Spain
Telephone: +34 93 581 2791
Fax: +34 93 581 2013
gregorio.alvaro@uab.cat
1

Authors’ e-mail addresses
a
Marie A. F. Delgove : marie.delgove@maastrichtuniversity.nl; Daniela Valencia:
b
danielapatricia.valencia@uab.cat; Jordi Solé : jordi.sole.ferre@uab.cat; Katrien V.
a
a
Bernaerts : katrien.bernaerts@maastrichtuniversity.nl; Stefaan M. A. De Wildeman :
b
s.dewildeman@maastrichtuniversity.nl; Marina Guillén : marina.guillen@uab.cat
Graphical abstract
Highlights

TmCHMO was successfully immobilized and co-immobilized with GDH-Tac for the
first time onto an amino functionalized support
2


A new process for the enzymatic oxidation of 3,3,5-trimethylcyclohexanone by a self-
sufficient co-immobilized cyclohexanone
dehydrogenase.
monooxygenase and
glucose


Co-immobilized biocatalyst was re-used up to 15 cycles reaching 83% of average
conversion and 11.2 mg product mg biocatalyst-1 of biocatalyst yield.
Co-immobilized biocatalyst allows cofactor regeneration and improves biocatalyst
yield compared to the soluble enzymes bringing closer their industrial implementation
in polymers industry.
ABSTRACT
The industrial application of Baeyer-Villiger monooxygenases (BVMOs) is typically
hindered by stability and cofactor regeneration considerations. The stability of biocatalysts
can be improved by immobilization. The goal of this study was to evaluate the (co)-
immobilization of a thermostable cyclohexanone monooxygenase from Thermocrispum
municipale (TmCHMO) with a glucose dehydrogenase (GDH) from Thermoplasma
acidophilum for NADPH cofactor regeneration.
Both enzymes were immobilized on an amino-functionalized agarose-based support (MANA-
agarose). They were applied to the oxidation of 3,3,5-trimethylcyclohexanone for the
synthesis of ε-caprolactone derivatives which are precursors of polyesters. The performances
of the immobilized biocatalysts were evaluated in reutilization reactions with as many as 15
cycles and compared to the corresponding soluble enzymes. Co-immobilization proved to
provide the most efficient biocatalyst with an average conversion of 83% over 15
3

reutilization cycles leading to a 50-fold increase of the biocatalyst yield compared to the use
of soluble enzymes which were applied in a fed-batch strategy.
TmCHMO was immobilized for the first time in this work, with very good retention of the
activity throughout reutilization cycles. This immobilized biocatalyst contributes to the
application of BVMOs in up-scaled biooxidation processes.
Keywords: biocatalyst immobilization, Baeyer-Villiger monooxygenase, lactone monomer,
cofactor recycling, glucose dehydrogenase
1
. INTRODUCTION
Enzymatic reactions have been identified as a sustainable technology since they usually
follow the rules of green chemistry.[1, 2] Oxidative biocatalysis, and Baeyer-Villiger
monooxygenases (BVMOs) in particular, is an alternative of lesser toxicity compared to
chemical oxidation.[3] BVMOs are biocatalysts capable of catalyzing the oxidation of
(cyclic) ketones by inserting one atom of oxygen in a C-C bond, therefore generating water as
by-product. BVMOs can catalyze the oxidation of a wide range of cyclic ketones of various
ring sizes including alkyl substituted ketones, as well as perform enantioselective
sulfoxidation.[4-6] These oxidative enzymes have been applied to the synthesis of
intermediates for the pharmaceutical industry,[7-9] and chiral molecules for fine chemical
and fragrances.[10] Additionally, several BVMOs have been identified as relevant
biocatalysts for the synthesis of lactones as monomers for polymeric materials, for example,
ε-caprolactone, either from whole-cell[11] or via a cascade reaction,[12] lauryl lactone,[13]
which is a nitrile-substituted ε-caprolactone used as precursor for polyamide,[14] and β,δ-
4

trimethyl-ε-caprolactone (TMCL).[15, 16] Alkyl substituted lactones are particularly
interesting for the synthesis of polyesters with low glass transition temperature (T < 0 °C in
g
general).[17] This property enables applications such as biodegradable plasticizers[18] or
encapsulating agents for coating formulations[19] with polymers from TMCL for example.
The applicability of BVMOs is however hindered by their lack of robustness, either due to
thermolability or to limited stability in the presence of organic solvents. Using protein
engineering, several mutants of cyclohexanone monooxygenase with improved
thermostability were created.[20-22] The discovery of new thermostable BVMOs contributes
to the development of their applicability in biotransformations.[23-26] Recently, a
cyclohexanone monooxygenase from Thermocrispum municipale DSM 44069 (TmCHMO;
EC 1.14.13.22) was identified as being particularly relevant for the preparation of lactones as
polymeric building blocks due to its high thermostability, good resistance to organic solvents,
and broad substrate scope towards cyclic ketones.[27, 28]
Although TmCHMO has already been applied for the synthesis of ε-caprolactone derivatives
from 3,3,5-trimethylcyclohexane, using either a self-sufficient fused biocatalyst[15] or a
glucose dehydrogenase to regenerate the NADPH co-factor,[16] this enzyme has not yet been
immobilized. Immobilization of whole-cells or isolated enzymes is indeed known to increase
the operational stability of enzymes. Additionally, immobilization has several advantages
including facilitating the recovery of the biocatalyst, decreasing the costs of downstream
processing, and potentially decreasing the enzyme cost per kilogram of product, provided that
the immobilized biocatalysts maintain their activity throughout the reuses.[29, 30]
So far, isolated BVMOs have mostly been immobilized to polymeric supports by covalent
binding.[31] For example, a cyclohexanone monooxygenase from Acinetobacter
calcoaceticus (AcCHMO) was immobilized on Eupergit (polyacrylamide based supported
5

beads) via covalent binding with a glucose 6-phosphate dehydrogenase for the synthesis of
chiral lactone building blocks.[32] Fusions of AcCHMO with a polyol dehydrogenase were
similarly immobilized for the synthesis of ε-caprolactone.[33] The immobilized biocatalyst,
however, displayed a low stability on the support and a poor operational stability. Recently,
MANA-agarose (monoaminoethyl-N-aminoethyl)-agarose was identified as a suitable
support for the immobilization of a fused AcCHMO-phosphite dehydrogenase (AcCHMO-
PTDH).[34] For this enzyme, a higher retained activity was achieved with metal-chelate
supports such as Ni-iminodiacetic acid (Ni-IDA) and Co-IDA.[34]
In this article, our goal is to expand the use of immobilized BVMOs and evaluate them for
the synthesis of lactones as polymeric building blocks. The immobilization of TmCHMO and
a glucose dehydrogenase from Thermoplasma acidophilum (EC 1.1.1.47) (GDH-Tac) are
described with the aim of oxidizing 3,3,5-trimethylcyclohexanone to alkyl substituted ε-
caprolactone derivatives (Figure 1). The enzymes were immobilized on a MANA-agarose
support, either separately or co-immobilized on the same support, by covalent bonding. In
total, three biocatalyst formats were examined for TmCHMO and GDH-Tac: soluble
enzymes, enzymes immobilized separately, and co-immobilized enzymes. The performances
of the immobilized enzymes were evaluated in over 15 repeated biooxidation cycles, in which
the immobilized biocatalysts are re-used each time, and compared to the corresponding
soluble enzymes.
2
2
. EXPERIMENTAL
.1.Chemicals.
3
,3,5-Trimethylcyclohexanone (98%, Sigma-Aldrich), methanol (Biosolve), (+)-glucose
(>99%, Alfa Aesar) were used as received. High-density aminoethyl 4BCL agarose (MANA-
agarose, Agarose Beads Technologies) was stored at 4 ˚C. β-Nicotinamide adenine
6

+
dinucleotide phosphate disodium salt (NADP , 97%, Alfa Aesar), and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, ≥97%, Sigma-Aldrich) were stored at -
2
0 ˚C. TmCHMO and GDH-Tac were produced and supplied by InnoSyn BV (Geleen, The
Netherlands).
2
.2.TmCHMO and GDH-Tac activity assays
TmCHMO activity in the CFE was determined spectrophotometrically following NAPDH
-1
-1
consumption at 340 nm (ƐNADPH = 6.22 mM cm ) with cyclohexanone as a substrate. The
mixture contained Tris-HCl (50 mM, pH 8.5), cyclohexanone (0.5 mM), NADPH (0.1 mM).
One unit of TmCHMO (U) was defined as the amount of enzyme required to catalyze the
+
conversion of 1 µmol of NADPH to NADP per min at 20 °C and pH 8.5.[34]
GDH-Tac activity was determined spectrophotometrically at 340 nm following the NADPH
-1
-1
(ƐNADPH = 6.22 mM cm , 400 µM) formation using D-Glucose (200 mM) as substrate and
sodium phosphate buffer 100 mM pH 8.0.[35] The basal production of NADPH by unspecific
enzymes present in the lysate was determined by this same test but avoiding the addition of
substrate and adding buffer instead of the amount of enzyme required. This production rate is
subtracted from the measurement with D-glucose. One unit of activity (U) was defined as the
+
enzyme required to convert 1 µmol of NADP per min at those given conditions (30 ºC, pH
8
.0). The absorbance was recorded using a spectrophotometer Cary 50 Bio UV-visible (Palo
Alto, USA).
2
.3.Preparation of immobilized TmCHMO and immobilized GDH-Tac
The general procedure for the covalent immobilization of the enzymes on MANA-agarose
-1
(
density: 1.07 g mL ) comprised three main steps: i) the ionic adsorption of the enzyme to
the support, ii) the addition of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) as an
activating agent to promote amide bond formation between the amino groups of the support
7

and the carboxyl groups of the enzyme (glutamic and aspartic residues), and iii) the addition
of NaCl to desorb all the enzyme that was not covalently bound to the support. After the
immobilization, the derivatives were washed carefully.
The immobilization of TmCHMO was carried out by suspending the support in 25 mM MES
buffer (pH 6.0); then the enzyme was added to the suspension and left to adsorb ionically to
the support for 0.25 h. After that time, EDC was added to final concentrations of 25 or 35
mM and left for 2 h. Finally, NaCl was added to a final concentration of 1 M and incubated
for 1 h. The immobilized derivative was washed with distilled water and filtered.
The immobilization of GDH-Tac was performed in 50 mM sodium phosphate buffer (pH
6
.0). The ionic adsorption step was completed after 0.5 h. A 200 mM stock solution of EDC
was prepared, the pH was adjusted to 6.0 with HCl; different volumes were added to get final
concentrations of 1, 3, 5, 10 or 15 mM and incubated for 1h. Afterwards, NaCl was added to
a final concentration of 0.5 M and incubated for 0.5 h. Lastly, the support was washed gently
with 100 mM sodium phosphate buffer (pH 8.0) and filtered.
For the co-immobilization of TmCHMO and GDH-Tac, the support was suspended in 50 mM
sodium phosphate buffer (pH 6.0); both enzymes were added and incubated 0.25 h. After the
ionic step was completed, EDC was added to final concentrations of 10 or 20 mM and
incubated 1 h. NaCl was added to a final concentration of 1M. The derivative was washed
with distilled water and filtered.
The characterization of the immobilization was carried out by measuring the activity of the
supernatant and the suspension throughout the entire process, in order to determine the
retained activity (Equation (1)) and immobilization yield (Equation (2)). TmCHMO and
GDH-Tac immobilized on MANA-agarose were stored at 4 °C prior to use.
8

Final suspension activity - Final supernatant activity
Initial supernatant activity
Retained activity (%)=
×100
(1)
Initial supernatant activity -Final supernatant activity
Initial supernatant activity
Immobilization yield (%)=
×100 (2)
2
.4.Determination of enzyme content
The cell lysate was pre-clarified by centrifugation (3220 g for 15 min.), and the total protein
content was determined by means of a Bradford Protein Assay Kit (Thermo Fisher Scientific,
Waltham, USA) using bovine serum albumin as standard.
Enzyme content was assessed using sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) (NuPage 12%, Invitrogen, USA) ran in a Mini-PROTEAN II
apparatus (BioRad, USA) following the protocol of Laemmli et al.[36] Low range protein
markers were used for subunit molecular mass determination. Gels were stained using
-1
-1
, 17% w v-1
Coomassie G250 colloidal stain solution (34% v v ethanol, 2% v v H
3
PO
4
NH
4
SO
4
and 0.066% Coomassie G250) and Image LABTM software (BioRad, USA) was used
for image processing.
2
.5. Determination of the reaction progress for biocatalyzed reactions using GC-FID
The substrate and product concentration were determined by GC-FID analysis in triplicate.
Aliquots of the reaction mixture (50 μL) were taken and diluted in acetonitrile (950 μL). The
sample was centrifuged using an Eppendorf centrifuge 5424 to remove precipitated protein
and analyzed by gas chromatography (GC-FID). The concentrations of substrate and lactones
were determined using calibration curves. GC-FID analyses were performed using a
Shimadzu GC-2010 Plus Gas Chromatograph with a hydrogen flame-ionization detector and
an SPB-1 capillary column (30 m × 0.25 μm × 0.25 mm inner diameter). For kinetics, the
9

following program was used: starting temperature of 60 ˚C maintained for 2 minutes,
-1
temperature increased to 200 ˚C at a heating rate of 15 ˚C min and then maintained at 200
C for 2 minutes, and temperature finally increased to 320 ˚C at a heating rate of 20 ˚C min-1
˚
and maintained at 320 ˚C for 2 minutes (sample injected at 250 ˚C, with a split ratio of 10,
μL injection volume). The following retention times were observed for kinetic samples
2
measured from the reaction mixture: 6.83 min for substrate 1, 9.25 min and 9.36 min for
lactones 1b and 1a (Figure 1).
2
.6.Reaction set-up and reaction conditions
The enzymatic reaction was controlled with a Metrohm 887 Titrino Plus titration apparatus.
The pH was monitored and adjusted to pH 8.0 by automatic addition of a solution of NaOH
(1 M). The reaction was performed in a double walled-glass and the temperature was
maintained to 30 °C. The reactions were performed in potassium phosphate buffer (25 mM),
at pH 8.0. The reaction was stirred at 500 rpm, and air was bubbled in the reaction volume at
-1
a rate of 8 mL min .
2
.7.Bioreaction with soluble TmCHMO and GDH-Tac biocatalysts
The reaction vessel was loaded with 10 mM of 3,3,5-trimethylcyclohexanone (47.4 μL), 250
+
-1
μM of NADP (5.9 mg), 350 mM of glucose, and 10% v v of methanol (3 mL) for a total
-1
reaction volume of 30 mL. The reaction was started by the addition of a 3.07% v v of
soluble TmCHMO (0.921 mL of CFE containing 32.1 mg TmCHMO) and 4.87% v v-1
soluble GDH-Tac (1.422 mL of CFE containing 65.8 mg soluble GDH-Tac). Protein content
and enzyme content of cell lysates was evaluated by means of a Bradford Protein assay kit
and SDS-PAGE, respectively, as described in section 2.4.
1
0

An additional 10 mM of substrate (47.4 μL) was added every hour until a total of 140 mM of
substrate.
2
.8.Reusability of immobilized TmCHMO and GDH-Tac biocatalysts
The reaction vessel was loaded with 10 mM of 3,3,5-trimethylcyclohexanone (47.4 μL), 250
+
-1
μM of NADP (5.9 mg), 30 mM of glucose, and 10% v v of methanol (3 mL) for a total
-1
reaction volume of 30 mL. The reaction was started by the addition of 5% v v of
-1
immobilized TmCHMO (20 mg TmCHMO g support, 1.605 g of supported enzyme
-1
corresponding to 32.1 mg TmCHMO) and 5% v v of immobilized GDH-Tac (29 mg GDH-
-1
Tac g support, 1.605 g supported enzyme corresponding to 46.5 mg GDH-Tac). The
substrate and product concentration were determined by GC-FID analysis in triplicate. At the
end of the reaction, the immobilized TmCHMO and immobilized GDH-Tac were filtered and
washed with buffer. New reaction medium containing 10 mM 3,3,5-trimethylcyclohexanone,
+
-1
2
50 μM NADP , 30 mM glucose and 10% v v of methanol was prepared; to which the
immobilized TmCHMO and immobilized GDH-Tac rinsed with buffer were added to start
the reaction. The supported enzymes were stored at 4 °C overnight after cycles 5 and 10.
2
.9.Reusability of the co-immobilized TmCHMO and GDH-Tac biocatalysts
The reactions were performed in a similar fashion as for the immobilized TmCHMO and
-1
GDH-Tac biocatalyst. The biocatalyst concentration was 5.4% v v (18.4 mg TmCHMO and
-
1
9
3
.1 mg GDH-Tac g support, 1.74 g of supported co-immobilized enzymes corresponding to
2.1 mg of TmCHMO and 15.83 mg of GDH-Tac).
3
3
. RESULTS AND DISCUSSION
.1.Biocatalyst immobilization on MANA-agarose support
1
1

The main goal was the oxidation of 3,3,5-trimethylcyclohexanone using the thermostable
TmCHMO (Figure 1). The NADPH cofactor was regenerated by applying GDH-Tac, which
uses glucose as a sacrificial cosubstrate. For this, both enzymes were evaluated in their
soluble form as well as immobilized on MANA-agarose (separately or co-immobilized).
Firstly, the immobilization of TmCHMO and GDH-Tac on MANA-agarose was studied
aiming to define the best conditions for the immobilization of the biocatalysts following two
approaches: separate enzyme immobilization and co-immobilization. Aiming to characterize
the immobilization processes these studies were performed at low activity loads to ensure no
mass transfer limitations once the enzyme is immobilized in the support (Table 1).
In order to obtain the highest immobilization yield and retained activity, the immobilization
of TmCHMO on MANA-agarose was studied. For covalent immobilization in MANA-
agarose, EDC has to be added once the protein is ionically adsorbed to promote its covalent
binding to the support which occurs between the amino groups of the support and the
carboxyl groups of the protein. In previous reported studies on immobilization of CHMO
from Acinetobacter calcoaceticus on MANA-agarose it was shown that 25 mM of EDC led to
high immobilization yields (98.5 ± 1.1 %) and retained activities (69.3 ± 7.3 %).[34]
Therefore, 25 mM was selected as EDC concentration to perform the immobilization of
TmCHMO. The results showed that the BVMO was completely adsorbed after 0.25 h.
However, lower values of immobilization yield and retained activity as expected were
obtained after EDC incubation (77.0 ± 0.7 % and 59.7 ± 0.1 %, respectively). The low
immobilization yield could indicate that the EDC concentration used was not enough to
ensure a complete covalent attachment of TmCHMO. Thus, the concentration of EDC was
increased up to 35 mM allowing an immobilization yield of 93.0 ± 0.4 % and a retained
1
2

activity of 62.4 ± 2.1 % (Table 1). Therefore, 35 mM was selected as the most appropriate
concentration of EDC for TmCHMO immobilization in MANA-agarose.
Regarding the immobilization of GDH-Tac, from previous reported results it was shown that
the enzyme was ionically adsorbed onto MANA-agarose after 0.5 h and 10 mM of EDC was
a suitable concentration for its covalent immobilization.[35] However, in the present work,
the GDH-Tac immobilization was studied at different concentrations of EDC trying to find
compatible conditions for the subsequent co-immobilization of GDH-Tac and TmCHMO.
1
0mM of EDC led to 78.7 ± 3 % of immobilization yield and 57.1 ± 2 % of retained activity.
Nevertheless, when higher concentrations EDC were tested (15 and 20 mM) the obtained
retained activities were 16 % and 17 % lower, respectively, indicating that GDH-Tac could
be deactivated at concentration higher that 10 mM of EDC. This negative effect of EDC on
enzyme activity has been already reported for other enzymes.[37] Therefore, the optimum
EDC concentration found for TmCHMO immobilization (35 mM) was not tested according
to the negative results obtained at 15 and 20 mM. Lower concentration of EDC (1, 3 and 5
mM) were also tested. Lower immobilization yields (37.0 ± 5.0, 44 ± 6.0 and 53 ± 1.0 %,
respectively) and lower retained activities (37.0 ± 4.0, 25.0 ± 4.0 and 56.0 ± 3.0 %,
respectively) were obtained probably due to an uncomplete covalent attachment of GDH-Tac.
Therefore, 10 mM was selected as the most appropriate concentration of EDC for this
enzyme immobilization in MANA-agarose (Table 1).
For the co-immobilization of TmCHMO and GDH-Tac, two EDC concentrations were tested,
1
0 and 20 mM. These values were selected taking into account the results obtained in the
previous immobilization studies carried out with the enzymes separately: concentration lower
than 10 mM were discarded due to the low expected retained activity of TmCHMO and
concentrations higher than 20 mM were not tested taking into account the loss of activity that
1
3

GDH-Tac suffers at high concentrations of EDC. Using 20 mM of EDC, the retained activity
of GDH-Tac was 19 % lower (38.9 ± 2.3 %) compared to the immobilization carried out at
1
0 mM of EDC and similar immobilization yield (98.7 ± 0.4 %) were obtained. Regarding
the TmCHMO, an increase from 10 to 20 mM did not improve significantly the obtained
immobilization yield. Finally, an EDC concentration of 10 mM was selected (Table 1) since,
as expected, GDH-Tac retained activity was significantly affected by high EDC
concentration.
3
.2.Reutilization of the immobilized biocatalysts and comparison with the soluble
enzymes
The soluble and immobilized biocatalysts were applied to the oxidation of 3,3,5-
trimethylcyclohexanone. Similarly to our previous studies with TmCHMO and this
substrate,[15] it was necessary to control the pH during the reaction since each molecule of
the substrate that was converted resulted in the formation of one molecule of D-
gluconolactone which spontaneously hydrolyzed to gluconic acid and consequently increased
the acidity of the reaction (Figure 1). Auto-titration of the reaction by addition of NaOH at 1
-1
M ensured a constant pH throughout the reaction course. A co-solvent (10% v v methanol)
was added to aid the solubility of the substrate, which is rather limited in water. This co-
solvent was selected based on our previous results showing that this co-solvent results in the
fastest reaction rate compared to other tested organic co-solvents.[15]
The oxidation of 3,3,5-trimethylcyclohexanone was first performed with both soluble
-1
TmCHMO and GDH-Tac using a TmCHMO load of 1.07 mg mL of reaction medium and
-1
an enzyme ratio of 1:2.0 (mg TmCHMO mg GDH-Tac ). These soluble enzymes are cell-
-1
-1
free extracts containing 34 mg of TmCHMO mL and 46 mg of GDH-Tac mL . The enzyme
concentrations were determined by means of Bradford assay kit and SDS-PAGE according to
1
4

the procedure described in section 2.4. Total conversion of the initial substrate (10mM) was
achieved in 1 h. Once the initial substrate was completely consumed, a fed-batch strategy was
applied by supplying an additional 10 mM of substrate to the reaction mixture every hour up
to a total of 150 mM of 3,3,5-trimethylcyclohexanone. The results showed that, while the
ketone was fully converted in 1 h for the first 3 substrate additions, the accumulation of
unreacted substrate was observed for the rest of the reaction until a final substrate
concentration of about 100 mM (Figure 2a). This change in the enzymatic reaction rate was
directly correlated to the amount of base needed to maintain the pH of the reaction, which is
related to the amount of gluconic acid co-product formed and substrate converted (Figure 2b).
The conversion for each addition was calculated and the obtained results are depicted in
Figure 3. A sharp decrease in conversion per substrate addition was observed until an average
conversion of about 10% was observed. This was attributed to the loss of enzymatic activity
during the reaction, but substrate inhibition of TmCHMO as a consequence of substrate
accumulation in the reaction mixture probably also played a role. Product accumulation in the
reaction media could also contribute to a decrease in subsequent conversions since BVMOs
often exhibit product inhibition, as has been previously reported by other authors.[31, 38, 39]
Process metrics were analyzed for the fed-batch strategy using soluble enzymes (Table 2).
The total process time after 14 additions was 14.4 h with a final product amount of 0.308 g
and a final unreacted substrate amount of 0.423 g. The biocatalyst yields reached 9.6 and 4.7
-1
mg of product mg of TmCHMO and GDH-Tac, respectively.
The performance of the TmCHMO and GDH-Tac which were separately immobilized at high
enzymatic loads was also studied. The TmCHMO immobilized derivative contained 20 mg of
-1
monooxygenase g of support, while the GDH-Tac derivative contained 29 mg of GDH-Tac
-1
g of support. Aiming to compare the results with the soluble enzymes, reactions with
1
5

separately immobilized enzymes were carried out using the same load of TmCHMO (1.07 mg
TmCHMO per mL of reaction). The ratio of TmCHMO/GDH-Tac was slightly lower (1:1.5)
-1
since it is determined by i) the maximum immobilized derivative that can be used (10% v v )
to ensure a proper suspension and mixing and, ii) the enzymes load per mg of support
obtained during the immobilization processes.
Separately immobilized derivatives were used in the biooxidation reaction, where the first
cycle took about 1.33 hour to total substrate conversion (Figure 4a). The increase in reaction
time for a total conversion of the substrate during the first cycle could be related to i) the
lower amount of loaded GDH-Tac with the immobilized enzymes which could lead to the
cofactor regeneration reaction being the limiting step or/and ii) diffusion limitations of the
NADP(H) co-factor between the bead particles containing TmCHMO and GDH-Tac or /and
iii) oxygen, glucose or 3,3,5-trimethylcyclohexanone mass transfer limitations due to the
diffusional restriction of these molecules in the support particles.
The operational stability of the biocatalysts was studied by re-using the same immobilized
enzymes over several reactions or cycles. At the end of the reaction, both immobilized
enzymes were recovered by filtrating the reaction mixture. These enzymes were reused in a
new reaction for conversion of the substrate, using the same reaction conditions for all cycles.
In total, the immobilized enzymes were reused up to 15 times aiming to compare the results
with the data obtained using soluble enzymes where 14 additions were carried out (Figure
4
b). Full conversion was obtained for the first 5 cycles, after which the conversion started to
decrease slowly.
The process metrics obtained using separately immobilized biocatalysts are shown in table 2.
Even though the total reaction time of the process was 1.4-fold higher, the average final
product amount (0.422 g) increased by 37 %. Moreover, the use of separately immobilized
1
6

enzymes also improves the process performance by reducing in 2.1-fold the final unreacted
substrate amount (0.199 g) and increasing the TmCHMO biocatalyst yield by 36%. The
overall biocatalyst yield is increased by 74% due to the better performances obtained with the
separately immobilized biocatalysts, despite the lower GDH-Tac biocatalyst loading (70% of
the GDH-Tac loading of the reaction with the soluble enzymes).
The performance of the enzymes that were co-immobilized at high loads was also studied
-1
-1
(
TmCHMO: 18.4 mg g of support; GDH-Tac: 9.1 mg g of support). In order to compare
the performance of the co-immobilized catalysts with the biocatalysts immobilized separately
and the soluble enzymes, the amount of co-immobilized support used in the oxidation
reaction was calculated so that the same amount of TmCHMO was applied in all cases (1.07
-1
mg mL ). The ratio TmCHMO/GDH-Tac in this case (1:0.5) was determined by the ratio
obtained during the co-immobilization process, where both enzymes compete for the same
support.
For this bioconversion, the reaction time was 1.17 h until the full conversion of the substrate,
1
7% higher compared to the soluble enzymes (Figure 5a). The higher reaction time compared
to the soluble enzymes could be due to the lower GDH-Tac load or to mass diffusional
restrictions, as already mentioned with the separately immobilized enzymes. However, even
though lower TmCHMO/GDH-Tac ratio was used when co-immobilized derivatives were
used (1:0.5) compared to the separately immobilized enzymes (1:1.5), the reaction time was
1
2% lower. Thus, the reduction of the reaction time of the co-immobilized derivative
compared to the separately immobilized biocatalyst probably indicates that NADP(H)
cofactor diffusional restrictions between bead particles is likely the main cause of reaction
time increase when separately immobilized derivatives are used.
1
7

The operational stability studies were also carried out with the co-immobilized derivative
during 15 cycles (Figure 5b). Compared to the biocatalysts immobilized separately, the co-
immobilized biocatalysts performed much better with the re-uses. A substrate conversion of
5
8% was achieved for the last cycle (15) compared to 39% substrate conversion obtained for
the same cycle with the biocatalysts immobilized separately.
Regarding the process metrics (Table 2), co-immobilization, in particular, proved to be the
best option of this biotransformation with higher average conversion over all re-utilization
cycles (83%) despite the lower concentration of GDH-Tac in the reaction. The highest
biocatalyst yields and final average product amounts were achieved with the co-immobilized
biocatalysts. Comparing to the separately immobilized enzymes, all process metric analyzed
were improved: 1.14-fold decrease in total process time, a 1.3-fold increase in final average
product amount, a 1.4-fold decrease in the unreacted substrate, a 1.1-fold increase in average
conversion, and a 1.3-fold increase in TmCHMO biocatalyst yield. The GDH-Tac biocatalyst
yield was improved by 3.7-fold because the experiment with the co-immobilized enzymes
achieved the best performances with the lowest GDH-Tac loading.
Compared to the soluble enzymes for which a fed-batch strategy was applied, even though
the total process time was slightly increased, the final average product formed was improved
in 1.7-fold, the unreacted substrate amount decreased in 3-fold, the average final conversion
was increased in 1.6-fold, and the total biocatalyst yield was 3.6-fold higher. These values
prove the better performance of the co-immobilized enzymes in the target reaction studied
compared to separately immobilized enzymes.
4
. CONCLUSIONS
TmCHMO was successfully immobilized on a MANA-agarose support with the co-enzyme
GDH-Tac to ensure co-factor regeneration. Both the enzymes immobilized separately and co-
1
8

immobilized displayed good retention of activity in repeated re-utilization for the oxidation of
,3,5-trimethylcyclohexanone. Co-immobilization proved to give the most efficient
biocatalyst format, achieving the highest average conversion over 15 re-utilization cycles
83%) and a high significant improvement of 3.6-fold of the total biocatalyst yield compared
3
(
to the soluble enzymes. Compared to the biocatalysts which were separately immobilized, a
highest reaction rate was observed which was attributed to more efficient diffusion of the
NADP(H) co-factor between the two enzymes immobilized on the same support.
Immobilization of TmCHMO with GDH-Tac was thus identified as being a relevant
biocatalyst format for lactone synthesis. This work demonstrates that immobilized
thermostable BVMOs are promising biocatalysts for the synthesis of lactones, and in
particular polymeric building blocks. Future work will aim at improving the process
performance metrics such as the volume of the reaction and the product concentration, which
are required for large-scale production of enzymatic reactions.
ACKNOWLEDGMENTS.
The research for this work has received funding from the European Union (EU) project
ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and
Innovation actions H2020-LEIT BIO-2014-1. The views and opinions expressed in this
article are only those of the authors, and do not necessarily reflect those of the European
Union Research Agency. The European Union is not liable for any use that may be made of
the information contained herein. The Department of Chemical, Biological and
Environmental Engineering of UAB constitutes the Biochemical Engineering Unit of the
Reference Network in Biotechnology (XRB), and the research group 2017-SGR-1462,
Generalitat de Catalunya. Daniela Valencia and Jordi Solé acknowledge UAB funding their
PhD grants.
1
9

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2
1

Figure 1. Biocatalyzed oxidation of 3,3,5-trimethylcyclohexanone 1 with TmCHMO and
GDH-Tac to give the regio-isomeric lactones 1a and 1b which can be polymerized by ring
opening polymerization. The enzymes were either immobilized on a MANA-agarose or
soluble.
2
2

Figure 2. a) Reaction course of the conversion of 3,3,5-trimethylcyclohexanone with soluble
TmCHMO and soluble GDH-Tac (TmCHMO/GDH-Tac 1:2.0) with the concentration of
substrate (blue circles) and product (black squares). The total amount of substrate
accumulated is shown with a pink dotted line. b) Profile of the volume of NaOH (1M) added
during the course of the reaction. The pink dotted line indicates the initiation rate of NaOH
addition. Reaction conditions: 10 mM of substrate initially + 10 mM every hour, 10% v v-1
-1
-1
-1
methanol, 3.07% v v soluble TmCHMO (1.07 mg mL ), 4.87% v v soluble GDH-Tac
-1
+
(
2.19 mg mL ), 350 mM glucose, 250 µM NADP . Error values correspond to standard
deviation (n=3).
2
3

Figure 3. Sequential additions of substrate for the reaction with soluble TmCHMO and
soluble GDH-Tac (TmCHMO/GDH-Tac 1:2.0) with conversion as a function of the number
of substrate additions (conversion = 1-([sub]
before the next addition of substrate and [sub]
addition of substrate). Error values correspond to standard deviation (n=3).
f
/[sub]
i
) with [sub] the substrate concentration
f
i
the substrate concentration after the last
2
4

Figure 4. Re-uses of TmCHMO and GDH-Tac immobilized on separate supports
TmCHMO/GDH-Tac 1:1.5) with a) reaction profile for cycles 1, 6, 10, 15; and b) substrate
(
conversion after 1.33 hour for all cycles. The vertical dotted lines indicate overnight storage
of the immobilized enzymes in buffer solution. Reaction conditions: 10 mM of substrate,
-1
-1
-1
1
0% v v methanol, 5% v v immobilized TmCHMO, 5% v v immobilized GDH-Tac, 30
+
mM glucose, 250 µM NADP , 1.33 h reaction time. Error values correspond to standard
deviation (n=3).
2
5

Figure 5. Re-uses of co-immobilized TmCHMO and GDH-Tac (TmCHMO/GDH-Tac 1:2.0)
with a) reaction profile for cycles 1, 6, 10, 15; and b) substrate conversion after 1.17 hour for
all cycles. The vertical dotted lines indicate overnight storage of the immobilized enzymes in
-1
-1
buffer solution. Reaction conditions: 10 mM of substrate, 10% v v methanol, 5.4% v v co-
+
immobilized TmCHMO and GDH-Tac, 30 mM glucose, 250 µM NADP , 1.17 h reaction
time. Error values correspond to standard deviation (n=3).
2
6

Table 1. Overview of the characterization of the immobilization of TmCHMO and GDH-Tac
on MANA-agarose under optimum conditions.
Enzyme
Offered enzyme
load*
Immobilization
yield (%)
Retained
activity (%)
U g support -1
-
1
TmCHMO
5 U g of support 93.0 ± 0.4
8 mg TmCHMO
62.4 ± 2.1
3.1 ± 0.1
(
-1
g of support)
-
1
GDH-Tac
5 U g of support 78.7 ± 3.0
3.7 mg GDH-Tac
57.1 ± 2.0
2.9 ± 0.14
(
-1
g of support)
7
(
9
(
9.4 ± 2.3
TmCHMO)
6.5 ± 0.4
GDH-Tac)
12.9 ± 1.6
(TmCHMO)
48.2 ± 6.5
(GDH-Tac)
0.7± 0.1
(TmCHMO)
2.4± 0.5
-1
Co-immobilized
TmCHMO and
GDH-Tac
5 U g of support
of each enzyme
(GDH-Tac)
Error values correspond to standard deviation (n=2)
*No substrate transfer limitations were found at this enzymatic load
2
7

Table 2. Overview of the performances of TmCHMO and GDH-Tac biocatalysts for the
oxidation of 3,3,5-trimethylcyclohexanone
Biocatalyst
format
Ratio
Total
Product Unreacted Average Biocatalyst yieldc
a
substratea conv
b
-1
TmCHMO reaction formed
(mg product mg biocatalysts )
:
GDH-Tac time
h)
(g)
(g)
(%)
(
TmCHMO
9.6 ± 1.2
GDH-Tac Total
(mg:mg)
Soluble
1:2.0
14.4
20.0
17.5
0.308 ±
0.423 ±
0.007
51 ± 3
73 ± 2
83 ± 1
4.7 ± 0.6
9.1 ± 0.7
3.1 ±
0.4
0
.040
Immobilized 1:1.5
0.422 ±
.034
0.199 ±
0.010
13.1 ± 1.0
16.8 ± 1.2
5.4 ±
0.4
0
Co-
immobilized
1:0.5
0.538 ±
0.039
0.138 ±
0.005
34.0 ± 2.5 11.2 ±
0.8
a
Cumulated amount of product and unreacted substrate (sum of each cycle for the
immobilized enzymes and value measured at the end of the reaction for the soluble enzymes)
bAverage conversion calculated for 15 cycles for the immobilized enzymes and for 14
c
-1
additions for the soluble enzymes. Biocatalyst yield = total mg of product mg of biocatalyst
TmCHMO, GDH-Tac or TmCHMO + GDH-Tac). Error values correspond to standard
deviation (n=3).
(
2
8