Toward Upscaled Biocatalytic Preparation of Lactone Building Blocks for Polymer Applications

Article abstract of DOI:10.1021/acs.oprd.8b00079

Although Baeyer-Villiger monooxygenases (BVMOs) have gained attention in recent years, there are few cases of their upscaled application for lactone synthesis. A thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) was applied to the oxidation of 3,3,5-trimethylcyclohexanone using a glucose dehydrogenase (GDH) for cofactor regeneration. The reaction progress was improved by optimizing the biocatalyst loading, with investigation into oxygen limitations. The product concentration and productivity were increased by keeping the substrate concentration below the inhibitory level via continuous substrate feeding (CSF). This substrate feeding strategy was evaluated against two biphasic reactions using either toluene or n-butyl acetate as immiscible organic solvents. A product concentration of 38 g L^-1 and a space-time yield of 1.35 g L^-1 h^-1 were achieved during the gram-scale synthesis of the two regioisomeric lactones by applying the CSF strategy. These improvements contribute to the large-scale application of BVMOs in the synthesis of branched building blocks for polymer applications.

Full text of DOI:10.1021/acs.oprd.8b00079

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Full Paper
Towards upscaled biocatalytic preparation of
lactone buildings blocks for polymer applications
Marie Delgove, Matthew Elford, Katrien Bernaerts, and Stefaan De Wildeman
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00079 • Publication Date (Web): 12 Jun 2018
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Towards upscaled biocatalytic preparation of lactone buildings blocks for polymer applications
Marie A. F. Delgove, Matthew T. Elford, Katrien V. Bernaerts, and Stefaan M.A.
De Wildeman*
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Affiliation:
Maastricht University, AachenꢀMaastricht Institute for Biobased Materials (AMIBM)
Brightlands Chemelot campus
Urmonderbaan 22, 6167 RD Geleen, The Netherlands
Correspondence: s.dewildeman@maastrichtuniversity.nl, +31 46 7022588
ABSTRACT
Despite BaeyerꢀVilliger monooxygenases (BVMOs) gaining attention in recent years, there are few cases
of their upꢀscaled application for lactone synthesis. A thermostable cyclohexanone monooxygenase from
Thermocrispum municipale (TmCHMO) was applied to the oxidation of 3,3,5ꢀtrimethylcyclohexanone,
using a glucose dehydrogenase (GDH) for coꢀfactor regeneration. Reaction progress was improved by
optimizing the biocatalyst loading, with investigation into oxygen limitations. Product concentration and
productivity were increased by keeping the substrate concentration below inhibitory level via continuous
substrate feeding (CSF). This substrate feeding strategy was evaluated against two biphasic reactions
ꢀ1
using either toluene or nꢀbutyl acetate as immiscible organic solvents. A product concentration of 38 g L
ꢀ1
ꢀ1
and a spaceꢀtime yield of 1.35 g L h were achieved during the gramꢀscale synthesis of the two regioꢀ
isomeric lactones by applying the CSF strategy. These improvements contribute to the largeꢀscale
application of BVMOs in the synthesis of branched building blocks for polymer application.
Keywords: reaction engineering, branched lactone, biocatalyst loading, product concentration, upꢀ
scaling, oxygen limitation
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INTRODUCTION
1
Oxidative chemistry plays a major role in the chemical industry. About one third of industrial
2ꢀ3
biocatalyzed processes involve redox enzymes, among half of which are oxidation reactions. These
biocatalysts offer an alternative of lower toxicity, replacing conventional toxic oxidants with molecular
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oxygen or H O , and by operating in mild aqueous conditions rather than in pure organic solvents.
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BaeyerꢀVilliger monooxygenases (BVMOs) in particular have gained interest in recent years, with efforts
6
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8
in enzyme discovery, enzyme engineering, and development towards their application. BVMOs are an
interesting alternative to the classical chemical BaeyerꢀVilliger reaction thanks to their enantioꢀ and
9
regioselectivity on a wide range of substrates. This differentiating feature is particularly relevant for
10
pharmaceutical compounds, where chirality is crucial. As a consequence, upscaling efforts at pilot plant
scale using BVMOs have mainly focused on pharmaceutical applications. For example, the oxidation of
racꢀbicyclo[3.2.0]heptꢀ2ꢀeneꢀ6ꢀone, whose oxidized product is a chiral building block for the synthesis of
prostaglandins, was performed at a 200 Lꢀscale using whole cells expressing a cyclohexanone
11
monooxygenase from Acinetobacter calcoaceticus NCIMB 9871 (AcCHMO; EC 1.14.13.22). BVMOs
have also successfully been identified as relevant industrial biocatalysts for the enantioselective
1
2ꢀ13
sulfoxidation of two key intermediates in the synthesis of pharmaceutical compounds.
The BVMOꢀ
14
catalyzed synthesis of a chiral sulfoxide drug intermediate was recently reported at kgꢀscale. Moreover,
BVMOꢀcatalyzed synthesis of lactones, which are relevant building blocks for polymers, has been applied
at smaller laboratory scale. For example, poly(εꢀcaprolactone) was synthesized from 6ꢀhydroxyhexanoic
15
acid prepared at multiꢀdozen gram scale in a fedꢀbatch biocatalyzed reactor. εꢀCaprolactone, which is
another precursor of this polyester, was prepared from a convergent cascade in a biphasic system
16
combining AcCHMO with an alcohol dehydrogenase (ADH). The same lactone was similarly obtained
ꢀ
by using whole cells expressing AcCHMO at 3 Lꢀscale, resulting in a moderate productivity of 0.79 g L
1
17
.
At the same scale, cyclododecanone was oxidized using recombinant E. coli whole cells expressing a
cyclopentadecanone monooxygenase from Pseudomonas sp. strain HIꢀ70 (PsCPDMO; EC 1.14.13) to the
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corresponding macrolactone. The multiꢀdozen gram scale synthesis of a nitrile εꢀcaprolactone
derivative, which is a Nylonꢀ9 precursor, was performed using PsCPDMO in whole cells with substrate
1
9
feeding product removal (SFPR).
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Several factors hinder however the application of BVMOs at industrial scale, namely poor stability
towards organic solvents, poor thermostability, and regeneration of their NAD(P)H coꢀfactor.
Thermostability and resistance to organic solvents can be improved by careful choice of the biocatalyst.
For example, a cyclohexanone monooxygenase from Thermocrispum municipale DSM 44069
(TmCHMO; EC 1.14.13.22) has recently been discovered, which outperforms most wildꢀtype CHMOs in
20
terms of thermostability with a melting temperature of 48 ˚C (see recent review on protein engineering
7
for a comparison of T ). This enzyme is particularly suitable for the application of BVMOs to the
m
synthesis of lactone polymeric building blocks given its broad substrate scope towards branched cyclic
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ketones. TmCHMO has already been engineered into a fusion protein, forming a selfꢀsufficient system
for NADPH coꢀfactor regeneration. For example, a TmCHMOꢀADH fusion biocatalyst enables the
22
cascade oxidation of cyclohexanol to εꢀcaprolactone. Recently, we have similarly applied a TmCHMOꢀ
PTDH fusion selfꢀsufficient biocatalyst, in which a phosphite dehydrogenase (PTDH) uses phosphite as
2
3
sacrificial coꢀsubstrate. Additionally, we have found that several water miscible coꢀsolvents are
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compatible with this enzyme, with good retention of substrate conversion. This enables increased
product concentration, which is typically achieved in successful biocatalyzed reactions operating at large
2
scale.
In this work, our goal is to optimize the TmCHMO biocatalyzed oxidation of 3,3,5ꢀ
trimethylcyclohexanone (Scheme 1). This substrate can be prepared by the hydrogenation of isophorone
which is obtained from the selfꢀcondensation of acetone, potentially sourced from renewables via the
24ꢀ26
acetoneꢀbutanolꢀethanol fermentation of lignoꢀcellulosic feedstocks.
The two regioꢀisomeric lactones
resulting from the oxidation of 3,3,5ꢀtrimethylcyclohexanone, i.e. β,δ,δꢀtrimethylꢀεꢀcaprolactone and
27ꢀ29
β,β,δꢀ trimethylꢀεꢀcaprolactone (TMCL), are attracting attention in the field of polymers.
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Scheme 1. Synthetic pathway to the substrate 3,3,5ꢀtrimethylcyclohexanone 1 (acetoneꢀbutanolꢀethanol
fermentation, acetone condensation to isophorone, isophorone hydrogenation) followed by biocatalyzed
BaeyerꢀVilliger oxidation to the regioꢀisomeric lactones 1a and 1b (TMCL). Two examples of polymers
derived from the polymeric building blocks TMCL including homopolymers, and copolymers with εꢀ
caprolactone and ωꢀpentadecalactone.
Using a selfꢀsufficient TmCHMOꢀPTDH fused biocatalyst, we have previously shown that the
biocatalyzed oxidation of 3,3,5ꢀtrimethylcyclohexanone suffers from substrate and product inhibition
2
3
(
Scheme 2a). This reaction leads to the formation of two regioꢀisomeric lactones. While BVMOꢀ
catalyzed oxidations favor the distal lactone, the chemical BaeyerꢀVilliger oxidation favors the formation
of the proximal lactone (55:45 and 45:55 1a:1b, respectively). With the fused TmCHMOꢀPTDH
ꢀ1
biocatalyst, a biphasic system in toluene yielded a maximum product concentration of 11 g L . Using the
same selfꢀsufficient biocatalyst, continuous feeding of substrate afforded a maximum productivity of 1.20
ꢀ1
ꢀ1
ꢀ1
g L h with a limited product concentration of 7 g L . In order to improve the TmCHMO catalyzed
oxidation of 3,3,5ꢀtrimethylcyclohexanone towards upscaling, high productivity and product
concentration were targeted while maintaining a low biocatalyst loading. To this end, TmCHMO was
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applied in a twoꢀenzyme system with a glucose dehydrogenase (GDH) to regenerate the NADPH coꢀ
factor using (+)ꢀglucose as coꢀsubstrate (Scheme 2b). Continuous substrate feeding (CSF) was selected as
substrate supply method in order to keep the substrate concentration below inhibitory level. The effects of
biocatalyst loading and oxygen input on the reaction progress were evaluated, since they can also increase
overall productivity. CSF was further optimized with varying feeding rate, total amount of added
substrate, and total reaction time. The best reaction conditions were subsequently evaluated against two
biphasic systems using either nꢀbutyl acetate or toluene as the waterꢀimmiscible organic phase. Finally,
the CSF biocatalyzed reaction was scaled to a total volume of 500 mL to yield the regioꢀisomeric lactones
at gramꢀscale.
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Scheme 2. Biocatalyzed oxidation of the branched substrate 3,3,5ꢀtrimethylcyclohexanone 1 to a mixture
of β,δꢀtrimethylꢀεꢀcaprolactone 1a and 1b with a) a fused TmCHMOꢀPTDH biocatalyst as shown in
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previous work, and b) a twoꢀenzyme system using a GDH to regenerate NADPH using (+)ꢀglucose as
sacrificial substrate.
MATERIALS AND METHODS
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Chemicals. 3,3,5ꢀtrimethylcyclohexanone (98 %, SigmaꢀAldrich), (+)ꢀglucose (> 99 %, Alfa Aesar),
sodium phosphite dibasic pentahydrate (> 98 %, Sigma Aldrich), hexadecane (99.5 %, TCI), methanol
(
(
Biosolve), nꢀbutyl acetate (99.5 %, Alfa Aesar), toluene (Biosolve), ethyl acetate (Biosolve) and Celite
Acros Organics) were used as received. βꢀNicotinamide adenine dinucleotide phosphate disodium salt
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+
NADP (97 %, Alfa Aesar) and glucose dehydrogenase GDHꢀ105 (Codexis) were stored at ꢀ20 ˚C and
solutions in phosphate buffer (25 mM, pH 8.0) were prepared fresh prior to use. Lactones 1a and 1b were
synthesized by chemical BaeyerꢀVilliger oxidation which was performed according to a procedure
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reported in the literature.
Recombinant production of TmCHMO in Escherichia coli. Cyclohexanone monooxygenase from
Thermocrispum muncipale (TmCHMO) was recombinantly produced in Escherichia coli in a 20 L batch
fermentor employing an E. coli K12 derivative and a pBAD/mycꢀHisC based expression vector. A
ꢀ
1
5
00 mL preꢀculture prepared in standard LuriaꢀBertani (LB) medium and supplemented with 100 µg mL
neomycin was used to inoculate 20 kg of main culture medium. The fermentation was performed using
terrific broth (TB) medium with glycerol. Preꢀsterilized Lꢀarabinose was added to the fermenter to a final
ꢀ1
concentration of 0.02 % w w after 2.5 h of inoculation. The cell material was harvested by centrifugation
4
h after inoculation of the fermenter, yielding 41 g wet cells per kg of fermentation broth. A cell free
extract of the protein was prepared by adding 2 weight equivalents of potassium phosphate buffer (100
mM, pH 7.0) to 1 weight equivalent of E. coli wet cells. The cell suspension was sonicated with an
ultrasound probe for 20 min with cooling on ice and then centrifuged to remove the cellꢀdebris. The
ꢀ
1
enzymatic activity for the CFE was determined in a spectrophotometric assay as being 0.12 U mg of
total CFE protein.
1
13
1
13
H and C NMR. Proton ( H) and carbon ( C) NMR spectra were recorded on a Bruker DPXꢀ300 MHz
1
apparatus at ambient probe temperature in CDCl . H NMR experiments were recorded with 32 scans and
3
13
C NMR experiments were recorded with 1024 scans. Chemical shifts are reported in ppm.
Heteronuclear single quantum coherence spectroscopy experiments (HSQC) were recorded with 4 scans.
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GC-MS. Gas chromatography coupled to mass spectrometry analyses were performed on a Shimadzu
GCꢀ2010 Plus Gas Chromatography system equipped with a Shimadzu GCꢀMSꢀQP2010 ultra mass
spectrometer detector and a SHꢀRxiꢀ5ms capillary column (30 m × 0.25 ꢁm × 0.25 mm inner diameter). A
mass/charge (m/z) range of 35ꢀ750 Da was analyzed. The temperature program was: starting temperature
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ꢀ1
of 60 ˚C held for 2 minutes, temperature increased to 200 ˚C with a heating rate of 15 ˚C min , and
ꢀ1
temperature finally increased to 320 ˚C at a heating rate of 20 ˚C min . A 2ꢀminute hold time was
implemented between each temperature increase.
GC-FID. Gas chromatography analyses were performed using a Shimadzu GCꢀ2010 Plus Gas
Chromatograph with a hydrogen flameꢀionization detector and a Supelco SPBꢀ1 capillary column (30 m ×
0
.25 ꢁm × 0.25 mm inner diameter). For kinetics, the following program was used: starting temperature
ꢀ1
of 60 ˚C maintained for 2 minutes, 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
ꢀ1
2
0 ˚C min and maintained at 320 ˚C for 2 minutes (sample injected at 250 ˚C, with a split ratio of 10,
2
ꢁL injection volume). The following retention times were observed for kinetics samples measured from
the reaction mixture: 6.58 min for the substrate 1, 9.18 min for the lactone 1b, and 9.29 min for the
lactone 1a. Isolated products after product recovery were measured with a modified method in which the
ꢀ1
temperature was increased from 60 °C to 200 °C at 10 ˚C min (sample injected at 300 ˚C, with a split
ratio of 100, 1 ꢁL injection volume). The following retention times were observed: 7.42 min for the
substrate 1, 11.16 min for the lactone 1b, 11.36 min for the lactone 1a, and 11.74 for the methyl esters 1a’
and 1b’.
Bioreactor set-up. Biocatalyzed reactions were performed with a Metrohm 877 Titrino plus titration
apparatus. Waterꢀsaturated air was bubbled through the reaction mixture with a Vögtlin redꢀy flow
controller. The pH was maintained at 8.0 by automatic titration of NaOH (1 M, Merck). In the case of
biocatalyzed reactions with a total volume of 30 mL, the reactor was a doubleꢀwalled vessel with a
volume of 90 mL and with the temperature kept constant at 30 °C. In the case of the biocatalyzed reaction
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with a total volume of 500 mL, the reactor was a doubleꢀwalled vessel with a volume of 1 L and with the
temperature kept constant at 30 °C. The air flow was provided with a sintered frit (1.3 cm diameter ×
2
cm length). The mixture was stirred with a IKA Eurostar Power control visc overhead stirrer equipped
with a 4ꢀblade glass propeller stirrer (5.5 cm diameter).
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Determination of the reaction progress for biocatalyzed reactions using GC-FID. Aliquots of the
reaction mixture (50 ꢁL) were taken and diluted in acetonitrile (950 ꢁL) containing hexadecane at a
concentration of 100 ꢁM as external standard. The sample was centrifuged, using an Eppendorf centrifuge
5424 to remove precipitated protein, and the supernatant was analyzed by GCꢀFID. Concentration of
substrate and lactones were determined using calibration curves.
Typical biocatalyzed reactions with continuous substrate feeding (CSF) at 30 mL scale. The reaction
+
ꢀ1
ꢀ1
vessel was loaded with NADP (250 ꢁM), glucose (375 mM), GDH (0.1 mg mL ) and 10 % v v
methanol, in phosphate buffer at 25 mM at pH 8.0. The reaction mixture was stirred at 500 rpm and air
ꢀ1
ꢀ1
was bubbled through at a rate of 16 mL min . The TmCHMO biocatalyst was added (1.5 mL, 5 % v v ).
Pure 3,3,5ꢀtrimethylcyclohexanone was fed continuously using a syringe pump (KDS Legato 110) with a
ꢀ1
feeding rate of 15 mM h for 16 hours for a total substrate volume of 1.138 mL (7.2 mmol, 240 mM).
Reaction progress was followed by GCꢀFID analysis. The reaction was stopped after 24 h by addition of
methanol (30 mL). The denatured protein was separated from the reaction mixture by centrifugation
(5000 rpm, 20 min), and filtration of the supernatant over Celite. Methanol was removed under rotary
evaporation, and the remaining aqueous phase was extracted with ethyl acetate (3 × 30 mL). The organic
phase was dried over MgSO and solvent was removed by rotary evaporation to afford a colorless oil
4
1
13
(
0.302 g, 32 % isolated yield). The GCꢀMS and H and C NMR spectral data were similar to the ones
23
we already reported in the literature.
Typical biocatalyzed reactions in aqueous-organic medium (biphasic reactions). The reaction vessel
+
ꢀ1
was loaded with NADP (250 ꢁM), glucose (375 mM), and GDH (0.1 mg mL ) in phosphate buffer at 25
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mM at pH 8.0. The reaction mixture was stirred at 250 rpm and air was bubbled through at a rate of
ꢀ1
1
6 mL min . 3,3,5ꢀTrimethylcyclohexanone (7.2 mmol, 1.138 mL) was dissolved in either toluene or nꢀ
butyl acetate (10 mL) and the organic phase was added to the aqueous phase. The TmCHMO biocatalyst
ꢀ1
was added (1.5 mL, 5 % v v ) to start the reaction. Reaction progress was followed by GCꢀFID analysis
of the aqueous and organic phase. The reaction was stopped after 26.5 hours by addition of the same
organic solvent (30 mL). The two phases were separated by centrifugation (5000 rpm, 30 min), and the
1
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organic phase was dried over MgSO and removed under rotary evaporation to afford a colorless oil
4
(toluene as the organic phase: 0.409 g, 25 % yield); (nꢀbutyl acetate as the organic phase: 0.432 g, 22 %
yield).
Typical biocatalyzed reactions with continuous substrate feeding (CSF) at 500 mL scale. The
+
ꢀ1
reaction vessel was loaded with NADP (250 ꢁM), glucose (375 mM), GDH (0.1 mg mL ), 3,3,5ꢀ
ꢀ1
trimethylcyclohexanone (0.394 mL, 5 mM), and 10 % v v methanol, in phosphate buffer at 25 mM at pH
ꢀ1
8
.0. The reaction mixture was stirred at 400 rpm and air was bubbled through at a rate of 30 mL min .
ꢀ1
The TmCHMO biocatalyst was added (25 mL, 5 % v v ). Pure 3,3,5ꢀtrimethylcyclohexanone was fed
ꢀ1
continuously using a syringe pump (KDS Legato 110) with a feeding rate of 15 mM h for 16 hours for a
total substrate volume of 18.912 mL (120 mmol, 240 mM). Reaction progress was followed by GCꢀFID
ꢀ1
analysis. Additional TmCHMO biocatalyst was added after 22 h (12.5 mL, 2.5 % v v ).The reaction was
stopped after 28 h by addition of methanol (500 mL). The denatured protein was separated from the
reaction mixture by centrifugation (5000 rpm, 20 min), washed with ethyl acetate (24 × 30 mL, 720 mL
of ethyl acetate in total), and separated by centrifugation (5000 rpm, 20 min). The ethyl acetate solution
was dried over MgSO , and the solvent removed under rotary evaporation to afford a yellow oil (0.364 g,
4
1
.6 % yield). The mixture as analyzed by GCꢀFID consisted of the desired products 1a and 1b (84.15 %),
the substrate 1 (11.73%), and the methyl esters 1a’ and 1b’ (4.12 %) (see Figure S3 for GCꢀFID analysis).
Methanol contained in the supernatant was removed under rotary evaporation, and the remaining aqueous
solution was filtered over Celite followed by saturation with NaCl. The aqueous solution was extracted
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with ethyl acetate (3 × 600 mL). The organic phase was dried over MgSO and the solvent was removed
4
by rotary evaporation to afford a yellow oil (13.0231 g, 64.9 % yield) which consisted of the desired
products 1a and 1b (93.0 %), the substrate 1 (0.2 %), and the methyl esters 1a’ and 1b’ (6.8 %), as
analyzed by GCꢀFID (see Figure 8b for GCꢀFID and Figure S4 for HSQC analysis).
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Kinetics of the formation of methyl esters 1a’and 1b’ (model reaction). A mixture of lactones 1a and
1
b prepared by chemical BaeyerꢀVilliger oxidation (1.055 mL) was dissolved in phosphate buffer at pH
8
.0, 25 mM (25.945 mL) and methanol (3 mL). Additional methanol was added (30 mL) and the reaction
mixture was left to react at room temperature, without stirring. The kinetics of the formation of the methyl
esters was followed on GCꢀFID. Samples for analysis consisted of 300 ꢁL of reaction mixture diluted in
700 ꢁL of acetonitrile.
RESULTS AND DISCUSSION
In order to increase the productivity of the TmCHMOꢀbiocatalyzed oxidation of 3,3,5ꢀ
trimethylcyclohexanone, several parameters were investigated, namely the biocatalyst loading, and the
oxygen input via the stirring rate and the air flow. For this reaction, control of pH is essential since GDH
was selected as coꢀenzyme to regenerate the NADPH coꢀfactor. In this case, for each molecule of
converted substrate, one molecule of gluconic acid is formed as a result of the spontaneous hydrolysis of
the coꢀproduct Dꢀgluconoꢀlactone, causing a drop in the pH throughout the reaction (Scheme 2b). The pH
of the reaction was therefore maintained at 8 by autoꢀtitration. Given the limited water solubility of the
ꢀ1
substrate (about 4 g L in buffer), the use of a coꢀsolvent is necessary to solubilize it at high
concentration. We have previously shown that the coꢀsolvent influences the reaction rate, with methanol
2
3
giving the fastest conversion. For this reason, methanol was selected as coꢀsolvent.
Biocatalyst loading
The stability of the TmCHMO biocatalyst was evaluated by performing bioconversions of decreasing
biocatalyst loading. These reactions were performed in a fedꢀbatch manner, meaning that about 10 mM of
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substrate was added stepwise every hour. This time interval was chosen to give the biocatalyst enough
time to fully convert the substrate, so that no substrate accumulation was observed. Under these hourly
substrate additions, the reaction progress of the bioconversions was found to be independent of the
ꢀ1
ꢀ1
decrease in the load of biocatalyst, from a range of 10 % v v to 1.25 % v v of TmCHMO (Figure 1 aꢀ
d). For all reactions, the substrate was fully converted within one hour, independent of the TmCHMO
concentration. This indicates good stability of the enzyme, at least within the time frame of the reaction.
Moreover, the product accumulation as measured in the aqueous phase followed the total amount of
substrate fed, indicating good efficiency of the enzyme. Interestingly, it was observed that the substrate
acted as antiꢀfoaming agent, with foaming only observed when there was no more substrate available in
the reaction mixture. As long as there was substrate, foaming was almost suppressed, preventing the
possible air/protein interphase pathway of protein deactivation taking place.
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8
9
0
Figure 1. Concentration of substrate and product as a function of time for increasing TmCHMO
biocatalyst loading. The arrows indicate batch additions of 10 mM of substrate. Reaction conditions:
ꢀ1
+
ꢀ1
initial [substrate] = 10 mM , 10 % v v methanol, [NADP ] = 250 ꢁM, [GDH] = 1 mg mL (except for a)
ꢀ1
ꢀ1
0
.1 mg mL ), [glucose] = 125 mM, air flow 8 mL min , stirring rate 500 rpm, total volume 30 mL.
ꢀ1
ꢀ1
The concentration of GDH was successfully decreased from 1 mg mL to 0.1 mg mL for the lowest
ꢀ1
TmCHMO concentration (1.25 % v v ), without negatively affecting either substrate conversion or
product accumulation. The biocatalyst loading can by consequence be decreased by a factor 10 for both
enzymes, TmCHMO and GDH, while maintaining full substrate conversion in the same reaction time.
Based on the full conversion of 10 mM of substrate observed for all biocatalyst loading, a substrate
ꢀ1
feeding rate of at least 10 mM h seems to be appropriate.
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Oxygen mass transfer limitations
In BVMOꢀcatalyzed oxidations, the oxygen supply is critical since molecular oxygen is one of the
reactants in the reaction. The solubility of molecular oxygen in aqueous solutions at atmospheric pressure
1
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30
is very low (about 0.3 mM), while it is usually higher in organic solvents. However, the oxygen
solubility in a mixture of 20 % methanol in water is not significantly increased compared to water only
30
(
0.34 mM). It is therefore important to identify oxygen mass transfer limitations since they can limit
reaction productivity, and therefore the overall process performances. The oxygen transfer rate is
influenced by the size of the gas bubble in the reaction mixture. The effect of stirring speed and air flow
rate, which are two factors impacting the amount of dissolved oxygen, was evaluated on the reaction
progress of the reaction (Figure 2a).
Figure 2. a) Substrate conversion, and b) product concentration, as a function of time with increasing
stirring rate and air flow. Reaction conditions: [substrate] = 30 mM (22 mM for the reaction with 8 mL
ꢀ1
ꢀ1
ꢀ1
+
min and 500 rpm), 2.5 % v v TmCHMO, 10 % v v methanol, [NADP ] = 250 ꢁM, [GDH] = 0.1 mg
ꢀ1
mL , [glucose] = 125 mM, total volume 30 mL.
ꢀ1
Doubling the air flow from 8 to 16 mL min resulted in the same increase in conversion as doubling the
stirring rate from 500 to 1000 rpm did. However, the stirring rate of 1000 rpm led to a significant amount
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of observed denatured protein which was attributed to the increased airꢀaqueous solution interface, which
is detrimental to the activity of the protein. Increasing the stirring rate and/or air flow resulted in similar
mass balance for all reactions, indicating that there was no removal of substrate or product from the
reaction mixture through the air stream (Figure 2b). The optimal reaction conditions for this reaction were
0
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ꢀ1
thus identified to be an air flow rate of 16 mL min with a stirring speed of 500 rpm.
Increasing product concentration with continuous substrate feeding
In order for the TmCHMOꢀbiocatalyzed oxidation to be competitive against classical chemical Baeyerꢀ
ꢀ1
Villiger oxidation, it is necessary to achieve high product concentrations, typically exceeding 10 g L .
Continuous substrate feeding (CSF) was therefore selected as a suitable substrate supply strategy in order
to keep substrate concentrations low, and thus avoid substrate inhibition. Potential product inhibition was
ꢀ1
evaluated by performing a reaction containing an initial 30 mM (approximately 4.7 g L ) of chemicallyꢀ
synthesized lactones (Figure 3). While the initial presence of 30 mM of product did not seem to hamper
product accumulation, it did however result in lower substrate accumulation, maybe as a result of better
solubility of the substrate in the aqueous phase due to the presence of the product. In these reaction
conditions, no product inhibition for product concentrations lower than 30 mM was observed.
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Figure 3. Concentration of substrate and product as a function of time in the absence of initial product
(
full symbols), and with an initial product concentration of 30 mM (empty symbols). Reaction conditions:
ꢀ1
ꢀ1
ꢀ1
+
CSF of 12.5 mM h , 2.5 % v v TmCHMO, 10 % v v methanol, [NADP ] = 250 ꢁM, [GDH] = 0.1 mg
ꢀ1
ꢀ1
mL , [glucose] = 125 or 375 mM, air flow 8 mL min , stirring rate 500 rpm, and total volume 30 mL.
1
1
1
1
1
1
1
1
1
1
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A comparable low substrate accumulation and high substrate conversion were obtained for feeding rates
ꢀ1
ꢀ1
of 10 mM h and 15 mM h (Figure 4a). Increasing the feeding rate resulted in faster product formation,
and consequently higher product concentration because of the higher total substrate amount to be
ꢀ1
converted (Figure 4b). In the case of this reaction, a reaction rate of 15 mM h was favored as the final
product concentration was higher with a similar conversion. Based on the reaction progress observed for
ꢀ1
various TmCHMO loading (Figure 1), an initial biocatalyst loading of 2.5 % v v was selected.
ꢀ1
ꢀ1
Figure 4. Effect of substrate feeding rate with CSF of substrate of 10 mM h and 15 mM h with a)
concentration of substrate (left axis) and substrate conversion (right axis), and b) concentration of
ꢀ1
ꢀ1
+
product. Reaction conditions: 2.5 % v v TmCHMO, 10 % v v methanol, [NADP ] = 250 ꢁM, [GDH] =
ꢀ1
ꢀ1
0
.1 mg mL , [glucose] = 125 or 375 mM, air flow 8 mL min , stirring rate 500 rpm, total volume 30 mL.
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ꢀ
1
When the substrate was fed at a rate of 15 mM h for 7h, little substrate accumulation was observed
(
Figure 5a). Feeding at such a rate overnight should allow for a total product concentration of about
ꢀ1
3
7 g L . This was however not the case as the reaction suffered from significant substrate accumulation,
ꢀ1
up to about 95 mM (about 15 g L ). As expected, increasing the reaction time increased the protein
denaturation observed. Interestingly, the same continuous overnight feeding of substrate resulted in
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0
ꢀ1
almost no substrate accumulation when the reactor was loaded with 5 % v v biocatalyst instead of 2.5 %
ꢀ1
v v with the conversion increasing from 42 % to 85 %. Despite high substrate conversion, the product
ꢀ1
concentration in the aqueous phase remained limited to 15 g L in both cases, even with the presence of
ꢀ1
1
0 % v v of methanol (Figure 5b). This was due to the product being located in the aqueous phase at a
ꢀ1
concentration up to 15 g L but also as a second phase in the reaction mixture, and to some minor extent
adsorbed onto the denatured protein.
Figure 5. Concentration of substrate and product as a function of time for bioconversions with CSF of
ꢀ1
ꢀ1
ꢀ1
substrate at 15 mM h a) for 7 h and for 16 h with 2.5 % v v TmCHMO, and b) with 2.5 % v v
ꢀ1
TmCHMO and 5 % v v TmCHMO for 16 h. Reaction conditions: [substrate] = 110 or 240 mM, 10 %
ꢀ1
+
ꢀ1
v v methanol, [NADP ] = 250 ꢁM, [GDH] = 0.1 mg mL , [glucose] = 125 or 375 mM, air flow 16 mL
ꢀ1
min , stirring rate 500 rpm, total volume 30 mL.
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ꢀ
1
The volume of the methanol coꢀsolvent was increased from 10 % to 20 % v v in order to attempt to
increase the solubility of the product in the aqueous phase, and therefore the overall product concentration
as measured in the aqueous phase (Figure 6). An increased concentration of methanol coꢀsolvent resulted
in a lower substrate accumulation and a better product formation according to the substrate feed. This is in
line with the reported robustness of TmCHMO towards methanol, since the enzyme can retain 96 % of its
1
1
1
1
1
1
1
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1
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2
0
conversion yield towards 2ꢀbutanone in the presence of 30 % methanol. Although the addition of more
methanol seemed to be beneficial to the product solubility, a coꢀsolvent volume of 10 % was selected for
further experiments in order not to complicate the product recovery procedure.
ꢀ1
Figure 6. Concentration of substrate and product as a function of time with 10 % or 20 % v v methanol.
ꢀ1
ꢀ1
+
Reaction conditions: CSF of 10 mM h , 2.5 % v v TmCHMO, [NADP ] = 250 ꢁM, [GDH] =
ꢀ1
ꢀ1
0
.1 mg mL , [glucose] = 125 or 375 mM, air flow 8 mL min , stirring rate 500 rpm, and total volume
3
0 mL.
Using CSF, the best reaction conditions for achieving high substrate conversion and high product
ꢀ
1
ꢀ1
concentration were thus obtained with a TmCHMO biocatalyst loading of 5 % v v , using 10 % v v
ꢀ1
methanol as coꢀsolvent, and a continuous feeding rate of 15 mM h for a total of 240 mM of substrate to
be converted.
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Continuous substrate feeding vs. biphasic systems in organic solvents
This improved CSF reaction was evaluated against another substrate supply strategy, namely a biphasic
system using either toluene or nꢀbutyl acetate. The objective of the waterꢀimmiscible organic phase is
twofold: first acting as a substrate reservoir and decreasing the substrate concentration in the aqueous
phase where the biocatalyst is present, and secondly serving as a product sink, solubilizing the formed
product and ensuring high product concentration. Toluene was selected because of the favorable solvent
resistance of TmCHMO towards this solvent (no significant decrease in activity in the presence of
0
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0
ꢀ1
23
3
3 % v v toluene for the oxidation of cyclohexanone at 30 ˚C). nꢀButyl acetate was selected as a lowꢀ
3
1
environmentalꢀimpact alternative. Additionally, both solvents have a low solubility in water which
should result in a clear partitioning of substrate and product, and a high boiling point circumventing any
potential evaporation that might occur over time.
ꢀ1
A biphasic system consisting of 33 % v v of organic solvent (i.e. 30 mL of aqueous phase with 10 mL of
organic phase) was loaded with 7.2 mmol of substrate (1.138 mL), corresponding to the same amount of
substrate that was converted in the optimized CSF system. In the biphasic systems, the substrate
concentration was 180 mM for the overall reaction mixture (240 mM if all the substrate was in the
aqueous phase and 720 mM if all the substrate was in the organic phase). During the reaction the substrate
was found almost exclusively in the organic phase along with the formed product (Figure S1 bꢀd vs. aꢀc).
It was however difficult to follow the reaction progress of each phase due to the significant emulsification
that occurred. It is therefore probable that the amount of substrate and product measured in the (lower)
aqueous phase was overestimated due to contamination with the (upper) organic phase, while the amount
in the organic phase was probably underestimated due to the emulsification. This is particularly visible
with the sudden drop of product concentration in the toluene phase between 23 and 25 h (Figure S1b),
which was due to samples of the reaction mixture containing a mix of both phases.
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At the end of the reaction (26.5 hours), the reaction mixture was diluted with additional organic solvent in
order to deactivate all remaining protein. This resulted in almost full solubility of the substrate and
product in the organic phase (> 98 %). Analysis of the isolated products revealed both biphasic systems
displayed lower substrate conversion than that of the equivalent CSF reaction, with 32 % and 43 % of
initial substrate recovered for the toluene and nꢀbutyl acetate systems, respectively. In contrast, the
products isolated from the improved CSF reaction contained only 4% of ketone, with a substrate
conversion of 85 % (Figure 7).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Composition of the isolated products as analyzed by GCꢀFID of the reactions performed with
ꢀ1
CSF at 15 mM h , a biphasic system in nꢀbutyl acetate or toluene. The percentage of substrate 1 found in
the isolated product is indicated. The ratio of lactone 1a:1b is 62:38 for the two biphasic reactions, and
6
0:40 for the CSF reaction.
Gram-scale preparation of branched lactones
The best reaction conditions obtained using CSF were scaled up to a 500 mLꢀscale reaction (reaction setꢀ
ꢀ1
up is shown in Figure S2). The substrate was fed at a rate of 15 mM h for 16 h, after which the reaction
was left running. Unlike with the equivalent 30 mLꢀscale experiment, a large amount of unreacted
substrate (27 % of the total amount of substrate fed) was present in the reaction after 19 h (Figure 5b vs.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
Figure 8a). This was attributed to significant protein denaturation occurring during the upꢀscaled reaction
ꢀ1
which might be due to increased gas/aqueous interphase. An additional 2.5 % v v of TmCHMO
biocatalyst was therefore added, resulting in instantaneous substrate conversion until almost all substrate
was fully converted (> 99 % conversion).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Scaledꢀup biocatalyzed reaction at 500 mL with a) concentration of substrate and product as a
ꢀ1
function of time with a substrate feed of 15 mM h ; and b) composition of the isolated products as
analyzed by GCꢀFID (0.2 % of ketone 1, 93.0 % of lactones 1a and 1b, and 6.8 % of methyl esters 1a’
and 1b’). Reaction conditions: 5 mM initial substrate concentration + 240 mM added at a rate of 15 mM
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
h , 10 % v v methanol, [TmCHMO] = 7.5 % v v (initial 5 % v v + 2.5 % v v added after 22 h),
+
ꢀ1
ꢀ1
[
NADP ] = 250 ꢁM, [GDH] = 0.1 mg mL , [glucose] = 375 mM, air flow 30 mL min , stirring rate
4
00 rpm, total volume 500 mL.
ꢀ1 ꢀ1
A spaceꢀtime yield (STY) of 1.35 g L h was obtained with the 500 mLꢀscale reaction, similar to the
STY obtained at the 30ꢀmL scale, despite the increased reaction time needed to convert the remaining
substrate after addition of TmCHMO (Table 1). This extra biocatalyst loading resulted in a lower
biocatalyst yield compared to the smaller reaction. It should be noted that the product concentration
ꢀ1
measured in the aqueous phase reached saturation at about 15 g L , and is therefore not representative of
ꢀ1
the real total product concentration of the reaction which was about 38 g L . Increasing the reaction
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volume resulted in an increase in isolated yield with over 12 g of isolated lactones product for the 500
mLꢀupscaled reaction. The STY was therefore also calculated based on the amount of isolated product. In
that case, increasing the reaction volume doubled the STY as a result of the higher substrate conversion
and higher isolated yield. Analysis of the denatured protein at the end of the reaction revealed that it
adsorbed both products and substrate, with a mixture consisting mainly of lactone products (85%) with
some substrate (12%) (Figure S3). The isolated product additionally contained low amounts of sideꢀ
products which were identified by NMR as being the corresponding methyl ester ringꢀopened products
(Figure 8b and Figure S4). A model reaction of chemically synthesized lactones in a reaction mixture of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
5
0:50 % v v buffer and methanol confirmed that this sideꢀproduct formation was induced by the dilution
of the reaction mixture with methanol to deactivate all remaining protein, and can be avoided by direct
product recovery at the end of the reaction (Figure S5).
Table 1. Process metrics of the biocatalyzed oxidation of 3,3,5ꢀtrimethylcyclohexanone with TmCHMO
using the CSF strategy at a scale of 30 mL and 500 mL.
a
b
c
Reaction Volume Time Conv
[product] STY
Biocatalyst Biocatalyst
Isolated Isolated
product yield
ꢀ1
ꢀ1
d
e
type
(mL)
(h)
(%)
Total (in
aqueous
phase)
(g L h )
loading
yield
ꢀ1
(gprod gcwwꢀ1
From solution
(from isolated
product)
(gcww L )
(g)
(%)
)
ꢀ
1
(
g L )
Biphasic
(nꢀbutyl
acetate)
Biphasic
f
f
g
g
30 26.5
57
68
n.d.
n.d.
(0.32)
25.0
0.52
0.57
0.258
22
30 26.5
(0.36)
25.0
25.0
37.5
0.287
0.302
25
32
69
(
toluene)
CSF
31.8
(15.5)
37.8
1.33
(0.42)
1.35
*
30
24
28
85
1.29
1.01
CSF
**
5
00
> 99
12.115
(16.8)
(0.87)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
5
6
a
b
Conversion calculated at the end of the reaction by GCꢀFID analysis. Product concentration in the
aqueous phase measured by GCꢀFID analysis. Total product concentration calculated from the substrate
ꢀ1
conversion as [product]total = [substrate]initial × conv × Mproducts, with Mproducts = 156 g mol and
ꢀ1 c
[substrate]initial in mol L . Spaceꢀtime yield calculated from total product concentration (STY =
[
product]total / reaction time) and spaceꢀtime yield calculated from isolated product in parenthesis (STY =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
ꢀ1
misolated product / reaction time / Vreactor). Calculated with a biocatalyst concentration of 0.5 g L cell free
cww
e
extract. Calculated from the total product concentration: biocatalyst yield = [product] / biocatalyst
total
f
g
loading. Calculated from the composition of the isolated products. Calculated with a biocatalyst
ꢀ1
concentration of 0.5 gcww L cell free extract from the isolated amount of product. Reaction conditions for
ꢀ1
+
ꢀ1
biphasic systems: 7.2 mmol substrate, 5 % v v TmCHMO, [NADP ] = 250 ꢁM, [GDH] = 0.1 mg mL ,
[glucose] = 375 mM, 30 mL aqueous phase, 10 mL toluene or nꢀbutyl acetate, 250 rpm, air flow 8 mL
ꢀ1
ꢀ1
ꢀ1
min ; for CSF reactions: [substrate] = 240 mM added at a rate of 15 mM h , 10 % v v methanol,
+
ꢀ1
*
ꢀ1
[
NADP ] = 250 ꢁM, [GDH] = 0.1 mg mL , [glucose] = 375 mM. 5 % v v TmCHMO, 500 rpm and air
ꢀ1
**
ꢀ1
ꢀ1
flow 16 mL min . 5 + 2.5 % v v TmCHMO, 400 rpm, 30 mL min , initial substrate loading of 5 mM.
CONCLUSIONS
The goal of this work was to optimize the TmCHMOꢀbiocatalyzed oxidation of 3,3,5ꢀ
trimethylcyclohexanone at small scale (30 mL reaction volume), for which the NADPH coꢀfactor was
regenerated by oxidation of (+)ꢀglucose by a GDH. The use of isolated enzyme allowed control of the
progress of the reaction. In particular, the TmCHMO biocatalyst loading could be reduced by almost a
ꢀ1
factor 10 using only 1.25 % v v of enzyme, without any loss of activity during fedꢀbatch experiments.
Similarly, the GDH concentration could be reduced by a factor 10 during the same fedꢀbatch experiments.
Possible oxygen limitation was investigated by changing the amount of dissolved oxygen present through
altering stirring speed and air flow rate. The fastest reaction progress was obtained with increased air flow
and reduced stirring rate, which also resulted in a favorable foaming reduction.
Continuous substrate feeding (CSF) was applied to keep the substrate concentration below inhibitory
ꢀ1
level. The feeding rate could be increased up to 15 mM h without undesirable substrate accumulation. A
substrate conversion of 85 % was reached by optimizing the biocatalyst loading, as well as the time of
ꢀ1
substrate feed, yielding a product concentration above 31 g L . Compared to our previous results with the
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fused TmCHMOꢀPTDH biocatalyst on this substrate, the product concentration was increased by a factor
23
4
when using TmCHMO with GDH. The improved reaction was further evaluated against two biphasic
systems in toluene and nꢀbutyl acetate, in which significant amounts of unreacted substrate were found.
This was attributed to premature protein denaturation induced by foaming and increased interphase
between the aqueous phase and the organic solvent.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
Finally, a product concentration of about 38 g L was achieved with the upscaling of the CSF reaction to
5
00 mL. It was necessary to increase the biocatalyst loading since unreacted substrate was observed after
feeding of 240 mM of substrate was stopped. In future, this can be circumvented by employing
continuous feeding of the biocatalyst throughout the reaction to ensure a constant enzymatic activity in
ꢀ1
ꢀ1
the reaction mixture. Nevertheless, a productivity of 1.35 g L h was achieved for the first gramꢀscale
TmCHMOꢀbiocatalyzed oxidation of 3,3,5ꢀtrimethylcyclohexanone. Although improved, the reaction still
requires development in terms of product concentration and spaceꢀtime yield in order to reach the
performances of large scale biocatalyzed oxidations. Further work will investigate this reaction to
demonstrate the viability of biocatalyzed oxidations for the synthesis of branched lactones for polymer
applications.
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 H2020ꢀ
LEIT BIOꢀ2014ꢀ1. 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 authors would like to thank
InnoSyn BV for the preparation of the TmCHMO biocatalyst, and Jan Brummund and Martin Schürmann
for fruitful discussions and advice.
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6
Supporting information. Reaction progress of biphasic systems, picture of the upscaled reaction setꢀup,
composition of chemicals adsorbed on denatured protein during upscaling reaction, HSQC spectrum of
isolated products of the upscaled reaction (identification of methyl esters ring opened products), kinetics
of the formation of the methyl ester ring opened products during product recovery.
0
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