Process Development through Solvent Engineering in the Biocatalytic Synthesis of the Heterocyclic Bulk Chemical ε-Caprolactone

Article abstract of DOI:10.1002/jhet.2595

For an alternative synthetic approach toward the heterocyclic industrial chemical ε-caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.

Full text of DOI:10.1002/jhet.2595

Month 2016
Process Development through Solvent Engineering in the Biocatalytic
Synthesis of the Heterocyclic Bulk Chemical ε-Caprolactone
Anna Reimer,a† Severin Wedde,a† Svenja Staudt,b Sandy Schmidt,c Diana Höffer,d Werner Hummel,a,e Udo Kragl,d
Uwe T. Bornscheuer,c and Harald Grögera,b
*
aFaculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
bDepartment of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Henkestr. 42, 91054 Erlangen, Germany
cInstitute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-
Str. 4, 17487 Greifswald, Germany
dInstitute of Chemistry, University of Rostock, Albert-Einstein-Str. 3A, 18059 Rostock, Germany
eInstitute of Molecular Enzyme Technology, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, Germany
*
E-mail: harald.groeger@uni-bielefeld.de
Additional Supporting Information may be found in the online version of this article.
†Both authors equally contributed to this work.
Received July 28, 2015
DOI 10.1002/jhet.2595
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
For an alternative synthetic approach toward the heterocyclic industrial chemical ε-caprolactone, which is based
on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydro-
genase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out
identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system
were found to proceed faster at both investigated substrate concentrations of 40 and 80 mM, respectively, compared
with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions
were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high com-
patibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic
reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on
the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might
be due to limitations of the permeation of, for example, the substrate through the cell membrane.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
[5,6] for the subsequent Baeyer–Villiger oxidation under formation
of ε-caprolactone. It is noteworthy that apart from molecular oxy-
gen (required for the second step), no other co-substrates are re-
quired because the cofactor for the first step (NADP+ being
transformed to NADPH) is recycled in the second step (by oxida-
tion of NADPH to NADP+ and simultaneous reduction of molec-
ular oxygen to water). This concept, for which also the
Bornscheuer group developed independently a similar process
[7], is graphically shown in Scheme 1.
Although we could achieve an excellent conversion
(>95%) for this biotransformation at substrate concentra-
tions of 20–60mM, elevated substrate concentrations led
to decreased conversion [3]. We identified inhibition as
well as deactivation effects of the involved organic compo-
nents (cyclohexanol, cyclohexanone, and ε-caprolactone)
as reasons. Recently, Bornscheuer et al. found an opportu-
nity to circumvent these inhibition and deactivation effects
at elevated substrate and product concentration by a reac-
tive in situ product removal through in situ oligomerization
The heterocyclic compounds ε-caprolactam and ε-caprolactone
belong to the most important industrial bulk chemicals because of
their need as monomers for the manufacture of polymer products
[1,2]. With respect to the latter one, its today’s favored industrial
production technology running on a multi-10.000 tons scale is
based on a Baeyer–Villiger oxidation of cyclohexanone utilizing
acetic peracid as oxidizing agent [1]. Besides limited selectivity,
however, hazardousness of the needed peracid reagent represents
a drawback. Recently, jointly with the company Enzymicals AG,
we reported a de novo approach toward ε-caprolactone, which
starts from the industrial basic chemical cyclohexanol and un-
dergoes a direct oxidation toward ε-caprolactone utilizing only mo-
lecular oxygen as a reagent in aqueous medium [3]. As a catalyst,
two enzymes were used, namely, an alcohol dehydrogenase
(ADH) from Lactobacillus kefir [4] for the initial dehydrogenation
of cyclohexanol to cyclohexanone and a cyclohexanone
monooxygenase (CHMO) from Acinetobacter calcoaceticus
© 2016 Wiley Periodicals, Inc.

A. Reimer, S. Wedde, S. Staudt, S. Schmidt, D. Höffer, W. Hummel,
U. Kragl, U. T. Bornscheuer, and H. Gröger
Vol 000
Scheme 1. Concept of a direct one-pot transformation of cyclohexanol
with air toward ε-caprolactone.
terms of sustainability, most of them are listed as “preferred”
or “usable” according to the Solvent Guide from the
pharmaceutical company Pfizer [within their Green Chemis-
try/environment, health and safety (EHS) program] [14].
When conducting the biotransformations and determining
the concentrations of substrate (cyclohexanol), intermediate
(cyclohexanone) and resulting product (ε-caprolactone),
several interesting results were obtained (Fig. 1). First,
of ε-caprolactone with an additional enzyme [lipase CAL-A
(lipase from Candida antarctica A)] [8]. As in the past for a
range of enzymes in general and other Baeyer–Villiger
monooxygenases [9,10], in particular, solvent engineering
turned out as a promising tool for process optimization, we
became interested in studying the impact of organic water-
immiscible solvents on the reaction course also for our
ε-caprolactone-forming target process. However, what makes
this search challenging is the fact that the CHMO is known as
a sensitive enzyme, which, for example, is deactivated rapidly
in the presence of a larger amount of water-miscible solvents
as demonstrated by Secondo and Fraaije et al. recently [9b].
For our purpose, we focused on the use of hydrophobic or-
ganic solvents with a high logP value (in particular exceeding
logP of 2) because of the following reasons: (i) expected sim-
ple phase separation, (ii) the second phase could serve as a
reservoir of the starting material cyclohexanol, the intermedi-
ate cyclohexanone and the product ε-caprolactone leading to
a decreased solubility of these components in the aqueous
phase and, thus, suppressing the observed negative impact
of these components on the CHMO enzyme, (iii) proven suit-
ability as co-solvents for other enzymes (or expected to be
suitable because of positive results with related solvents for
such other enzymes) [11], thus making its application also
for the CHMO from A. calcoaceticus promising. Accord-
ingly, we studied the influence of different organic solvents,
and in the following, the results, revealing isooctane as most
feasible organic co-solvent among them for this biotransfor-
mation, are described.
Figure 1. Screening of aqueous-organic reaction media. [Color figure can
be viewed in the online issue, which is available at www.
wileyonlinelibrary.com]
whereas most of organic solvents gave a decrease of conver-
sion, it is noteworthy that with isooctane as a highly hydro-
phobic solvent, a conversion in the same range (and even
being slightly superior) to the one achieved when using free
buffer was detected. Second, in contrast, some of the sol-
vents such as ethyl acetate and toluene led to a more rapid
deactivation. Our hypothesis is that because of the high logP
for isooctane, in this case, solubility of the organic solvent in
water is low thus leading to a low or negligible deactivation
of the enzyme. Nevertheless, it is remarkable that the pres-
ence of the second liquid phase does not deactivate the
enzyme (as this is true for other cases as observed in the past
RESULTS AND DISCUSSION
In our initial solvent screening, we chose ethyl acetate
[EtOAc; logP 0.73 ( 0.05)] [12], methyl tert-butyl ether
[logP 0.94 ( 0.30)] [12], toluene [logP 2.73 ( 0.10)] [12],
methylcyclohexane [logP 3.88 ( 0.40)] [12], n-heptane
[logP 4.5 ( 0.25)] [12], and isooctane (logP 4.5) [13] as sol-
vent components because of their frequent use in organic
synthesis as standard extraction solvents. Furthermore, in
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Solvent Engineering in the Biocatalytic Synthesis of ε-Caprolactone
[11], e.g., for oxynitrilases and alcohol dehydrogenases).
Other highly hydrophobic solvents with the same or similar
logP value, such as heptane, gave less satisfactory results.
Being surprising on the first glance, this result might also un-
derline that other physicochemical properties play an impor-
tant role as well. For example, in spite of logP values being in
the same range, the solubility of water is significantly less in
isooctane (0.56mg/L, 25°C) compared with heptane (0.05g/
L, 20°C) [15], which might contribute to a higher stability of
the enzyme in case of the aqueous-isooctane biphasic
system.
Having in hand a promising co-solvent candidate with
isooctane, next we got interested in a more detailed insight
into the kinetics of this process as well as a comparison of
the reaction courses in the presence and absence of this co-
solvent isooctane. The reaction courses of these two pro-
cesses, which were again carried out at a substrate
concentration of 40 mM, are shown in Figure 2 (for these
experiments, the enzyme activities were measured prior
to use: alcohol dehydrogenase from Lactobacillus kefir
(Lk-ADH) crude extract/glycerol 1:1 (v/v) 48.08U/mL;
CHMO lyophilized crude extract gave significantly lower
activities (0.08 U/mg) than stated for the commercial sam-
ple (0.35 U/mg); thus, the amount of added enzyme (in
milligram) was accordingly higher). In both cases, a
quantitative conversion was reached after a sufficient re-
action time (23 h). However, a further interesting insight
into the impact of isooctane as a co-solvent one can be
gained when looking into the kinetics. Whereas in pure
aqueous buffer, initial conversion after 1 h was 8%,
followed by 20%, 34%, and 50% after 2, 3, and 4 h, re-
spectively, the conversions in the presence of 10%(v/v)
of isooctane were significantly higher at all stages of
the reaction course (before reaching a quantitative con-
version). After only 1 h reaction time, already 13% con-
version was obtained, which rapidly increased to 34%,
58%, and 82% after 2, 3, and 4 h, respectively. These re-
sults clearly indicate a beneficial impact when using 10%
(v/v) of isooctane as a co-solvent.
In addition, when increasing the substrate concentration
up to 80mM while keeping the overall amount of enzyme
unchanged, we observed a similar tendency of the reac-
tion course. The kinetic reaction courses of these reactions
at 80mM substrate concentration are shown in Figure 3
(for comparison, data for analogous reactions at 40mM
substrate concentrations are added in this figure). When
conducting the reaction in the presence of 10%(v/v) of
isooctane as a co-solvent, reaction proceeds faster,
reaching 11%, 27%, 42%, and 60% after 1, 2, 3, and
4 h, respectively. After 23 h reaction time, full conversion
was observed. Although full conversion was also ob-
served in the absence of isooctane in pure aqueous buffer,
reaction proceeds much slower with 5%, 14%, 22%, and
33% after 1, 2, 3, and 4h, respectively. It should be added
Figure 2. Biotransformations of 40 mM cyclohexanol with commercial
BVMO enzyme in the absence and presence of isooctane [10%(v/v)]
[Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
that preliminary studies showed a similar reaction course
when using an increased volume of 30%(v/v) of the co-
solvent isooctane (data not shown). From an industrial
point of view, recycling of the enzymes (e.g., by direct
re-use of the aqueous phase bearing the enzymes) is of
economic interest. Thus, studying the impact of isooctane
on the long-term stability of the enzymes is a task for pro-
spective experiments.
Next, we were interested about the impact of the “enzyme
formulation” on the reaction course, in particular about the in-
fluence of the use of the Baeyer–Villiger monooxygenase as
isolated enzyme or integrated in a whole-cell catalyst on the re-
action course. Thus, we prepared whole-cells of Baeyer–
Villiger monooxygenase (7.6 mU/mgwcw), and as a benchmark
reaction, we conducted the biotransformation using disrupted
cells and the resulting free enzymes (in combination with
non-soluble cell materials such as disrupted cell wall pieces).
When conducting the reaction with this prepared crude extracts
containing cell material, we obtained a slightly lower [com-
pared with the commercial Baeyer-Villiger monooxygenase
(BVMO), see Fig. 3] but still satisfactory reaction course, lead-
ing to a conversion of 78% after a reaction time of 23 h (Fig. 4;
conversion after 1, 2, 3, and 4h: 4%, 8%, 12%, and 17%).
However, when utilizing the whole-cells collected directly from
the fermentation broth (and although adding the same amount
of external cofactor as in the processes with the “free” en-
zymes), a dramatic decrease of conversion was observed, and
even after 23 h reaction time, only 7% conversion was found
(Fig. 4; conversion after 1, 2, 3, and 4 h: 1%, 1%, 2%, and
2%). Thus, permeation processes through the cell wall and
the cell membrane, and thus limitation of the transport of sub-
strate into the cell as well as accumulation of substrate, interme-
diate, and product in the cell (thus causing to an increasing
extend inhibition and deactivation effects), might play a role.
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A. Reimer, S. Wedde, S. Staudt, S. Schmidt, D. Höffer, W. Hummel,
U. Kragl, U. T. Bornscheuer, and H. Gröger
Vol 000
Figure 3. Biotransformations with recombinant BVMO in the presence
of isooctane [10%(v/v)] at substrate concentrations of 40 and 80 mM (for
comparison, the reactions course of the analogous reaction using 40 mM
substrate concentration as given in Fig. 2 is shown again here in Fig. 3;
for reasons of a better clarity, measured points are connected by lines).
[Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
Figure 4. Biotransformations with lysed cells and resting whole-cells
bearing the BVMO in recombinant form in the presence of isooctane
[10%(v/v)]. CHMO, cyclohexanone monooxygenase. [Color figure can
be viewed in the online issue, which is available at www.interscience.wi-
ley.com.]
EXPERIMENTAL
Thus, currently the favored process option is based on utiliza-
tion of free enzymes rather than whole cell catalysts, and im-
provement of whole-cell catalysis represents a task for future
work.
Screening of aqueous-organic reaction media (according to
Fig. 1).
In a 10-mL flask, the crude extract of CHMO
from A. calcoaceticus (3.82U) is dissolved in a solvent
mixture and well shaken. Cyclohexanol (1, 40mM referring
to the overall volume of solvent, consisting of aqueous and
organic phase), MgCl2 (1mM), and ADH from L. kefir
[40U, crude extract/glycerol 1:1(v/v)] are added. The
mixture is stirred for 20min at room temperature (RT). The
reaction is started by the addition of NADP+ (1 mol%,
1.57mg) and stirred for 24 h at RT. KPi-buffer (phosphate
buffer, pH7, 50 mM) with added organic solvent is used as
solvent mixture. The reaction mixture is distributed on seven
reaction vessels (Eppendorf AG, Hamburg, Germany) with
0.7mL each and treated with 0.7mL DCM
(dichloromethane). After 30-min shaking in a Thermomixer
(Eppendorf), the two-phase system is separated by
centrifugation at 13000rpm for 5min. The lower organic
phase is removed, and the remaining aqueous phase and the
denatured enzymes are treated with DCM again, and the
extraction is repeated two times. The organic phases are
collected in a 50-mL volumetric flask, which is filled up to
the calibration mark. Three probes are bottled, and the
conversion is determined via gas chromatography. For the
determination of the conversion via nuclear magnetic
resonance (NMR), the solvent of the collected organic
phases is evaporated at 900 mbar and 40°C bath
temperature. The entire reaction mixture is transferred into
an NMR-tube together with a freshly prepared urotropine
solution (0.17 M in CDCl3, 0.1mL) as well as CDCl3
(0.7mL). The conversion is determined by comparison with
the intensity of the singlet peak of the standard urotropine.
CONCLUSION
In this study, we conducted a solvent engineering for our
recently developed alternative synthetic approach toward
the heterocyclic industrial chemical ε-caprolactone, which
is based on a biocatalytic oxidation of readily available
cyclohexanol with air in aqueous media using an alcohol de-
hydrogenase and a Baeyer–Villiger monooxygenase as en-
zyme components. After identifying isooctane as a suitable
co-solvent, biotransformations were conducted in an
aqueous-isooctane biphasic solvent system. It is noteworthy
that these oxidation processes proceed faster at both investi-
gated substrate concentrations of 40 and 80mM, respectively,
compared with the analogous enzymatic reactions in pure
aqueous medium. In all cases, quantitative conversions were
observed after a reaction time of 23 h. The achievements in-
dicate a high compatibility of isooctane [10%(v/v)] with the
enzymes as well as the potential for an in situ removal of
the organic reaction components, thus decreasing inhibition
and/or destabilization effects of these organic components
on the enzymes used. Further process development of this
novel oxidation process for the synthesis of ε-caprolactone
is currently in progress. In contrast, so far the use of recombi-
nant whole-cells gave less satisfactory results, which might
be because of limitations of the permeation of, for example,
the substrate through the cell membrane.
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Solvent Engineering in the Biocatalytic Synthesis of ε-Caprolactone
Biotransformations in the absence and presence of [10%(v/v)]
of isooctane (according to Figs. and 3).
of E. coli BL21(DE3) (New England Biolabs GmbH,
Frankfurt am Main, Germany) containing the plasmid
encoded gene of the CHMO from A. calcoaceticus
2
The
biotransformations were performed in a 10-mL round-bottom
flask with 5mL aqueous reaction volume at 25°C in a
tempered water bath on magnet stirrer (IKA-Werke, Staufen,
Germany). Cyclohexanol (40 or 80 mM) was dissolved in an
aliquot of KPi-buffer (pH 7, 50 mM, 200 mg/L MgCl2). In
addition, 0.002 mmol NADP+ and CHMO from A.
calcoaceticus [3.82 U of lyophilized crude extract supplied by
Enzymicals AG (Greifswald, Germany); the enzyme activity
is referred to cyclohexanone in KPi-buffer (pH 7, 50mM,
200mg/L MgCl2) and was measured prior to use
spectrophotometrically; because significantly lower activities
were measured (0.076U/mg) than stated for the commercial
sample, a higher amount of enzyme (in milligram) has to be
added compared with the amount calculated based on the
given enzyme activity (0.351 U/mg) of the commercial
sample] were dissolved in the KPi-buffer. ADH from L. kefir
[40U crude extract/glycerol 1:1(v/v) recombinantly
overexpressed in Escherichia coli as previously described [4];
the enzyme activity is referred to acetophenone as reference
substrate in KPi-buffer (pH 7, 50mM, 200mg/L MgCl2)] was
added and filled up to 5mL aqueous reaction volume with
KPi-buffer. The solution was mixed by pipetting up and down
several times, and during the reaction, a 1.5-cm stirring bar
mixed the solution by rotating 340 rpm. In the experiments
with organic solvent, 0.5 or 1.5mL isooctane (corresponding
to 10%(v/v) and 30%(v/v), respectively) was added. After 1,
2, 3, 4, and 23 h, an aliquot of 0.5mL of the reaction mixture
was taken and extracted three times with 0.5 mL ethyl acetate
(with 2mM acetophenone as an external standard) by mixing
on a vortexer (Scientific Industries, Bohemia, NY) for 1min
at maximum speed and phase separation for 1min at 21.500g
in a microcentrifuge (VWR International GmbH, Darmstadt,
Germany). The organic phases were combined and filled up
to 2 mL with ethyl acetate (2mM acetophenone) in a
volumetric flask. After mixing, the liquid was dried with a
spatula tip of sodium sulfate (anhydrous). The samples were
(GenBank: BAA86293.1
– cloning was described
previously [8]) was used for inoculation of 30mL
lysogeny broth (LB) medium (10 g/L peptone, 5g/L yeast
extract, 10g/L NaCl) [16] containing 50 μg/mL
kanamycin. Cultivation was performed in a 300-mL
shaking flask with baffles at 37°C on an orbital shaker
(Infors GmbH, Einsbach, Germany) at 140 rpm overnight.
The overnight culture was used as an inoculum to adjust
200 mL terrific broth medium containing 50 μg/mL
kanamycin to OD600 0.05. Cultivation was performed in
a 1-L shaking flask with baffles at 37°C and 140rpm.
CHMO expression was induced between OD600 0.6 and
0.8 with 0.05mM Isopropyl β-D-thiogalactopyranosid
(ITPG). Cultivation was continued at 25°C for 5 to 6h.
Cells were harvested by centrifugation at 4.000 g for
10min at 4°C and washed twice within KPi-buffer (pH7,
50mM, 200 mg/L MgCl2). Without any freeze-thawing
step, the obtained cell pellet was adjusted with KPi-buffer
to 250 gwcw/L (wcw = wet cell weight).
Determination of the enzyme activity (of the Baeyer–Villiger
monooxygenase and alcohol dehydrogenase, respectively).
Enzyme activity was measured (in analogy to
standard protocols reported, e.g., in reference [17])
spectrophotometrically at 340 nm by consumption of
NADPH through oxidation toward NADP+ with a
spectrophotometer V630 (JASCO Germany GmbH,
Gross-Umstadt, Germany). Four hundred and forty
microliters KPi-buffer (pH 7, 50 mM, 200 mg/L MgCl2)
were mixed with 500 μL cyclohexanone (20 mM in KPi-
buffer) for CHMO activity determination or with 500 μL
acetophenone (22 mM in KPi-buffer) for ADH activity
measurement. After performing a blank measurement,
50 μL NADP+ (8 mM in KPi-buffer) were added under
rigorous mixing. The reaction was started by the addition
of 10 μL enzyme solution under rigorous mixing. The
slope (ΔE) of the observed linear range of minimal 60 s
was used for activity calculation using the following
equation:
analyzed by gas chromatography using
A GC-2010
(Shimadzu GmbH, Duisburg, Germany) with a BP5MS-
column (SGE Analyticals Science, Victoria, Australia) with
the following temperature profile: starting with a plateau
at 70°C for 3.8 min followed by heating up to 200°C with
40°C/min (SPLI1: 230°C; flow control: linear velocity;
linear velocity: 46.9; split ratio 100; injection volume: 1 μL;
FID: 230°C). Retention times of the analyzed compounds
were as follows: cyclohexanol 3.2min, cyclohexanone
3.3 min, acetophenone 5.5min, ε-caprolactone 6.0 min and
6-hydroxyhexanoic acid 6.8min. The conversion was
determined by relative analysis as it has been shown that
cyclohexanol and ε-caprolactone were extracted in the
identical ratio.
A ¼ ðΔEꢀVꢀf Þ=ðεꢀvꢀdÞ
with the enzyme activity A (U/mL), the time depending
change in extinction ΔE (1/min), the total volume V (mL),
the dilution factor f, the extinction coefficient ε of NADPH
at 340 nm [6.300 L/(mol·cm)], and the layer thickness
d (cm).
Biotransformations with whole-cells in the presence of [10%
(v/v)] of isooctane (according to Fig. 4). The experimental
setup was identical to the biotransformations described
previously with the exception of using lysed or intact
(unfrozen) resting E.coli whole-cells over-expressing
CHMO from A. calcoaceticus instead of commercially
Preparation of whole-cells containing a Baeyer–Villiger
monooxygenase in recombinant form.
A glycerol stock
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A. Reimer, S. Wedde, S. Staudt, S. Schmidt, D. Höffer, W. Hummel,
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Vol 000
supplied CHMO crude extract. For the determination of the
CHMO activity of the whole-cells, first, an aliquot of the
whole-cell catalyst was lysed. After cell lysis, the solution
was centrifuged at 4.000g for 10min at 4°C. An enzyme
activity measurement was spectrophotometrically performed
with the crude extract of the supernatant as described
previously. As the measured activity of free enzyme (U/mL)
correlates with the engaged cell concentration (250 gwcw/L),
one can examine the volume of the cell suspension that is
needed to achieve 3.82 U.
J Chem Soc Perkin Trans I 1996 755; (c) Lee, W.-H.; Park, Y.-C.; Lee,
D.-H.; Park, K.; Seo, J.-H. Appl Biochem Biotechnol 2005, 121–124, 827.
[6] This enzyme is available in commercial form from Enzymicals AG
and such a commercial sample has been used in most experiments of our study,
for more information about this BVMO, see: http://www.enzymicals.de
[7] Mallin, H.; Wulf, H.; Bornscheuer, U. T. Enzyme Microb
Technol 2013, 53, 283.
[8] (a) Schmidt, S.; Scherkus, C.; Muschiol, J.; Menyes, U.;
Winkler, T.; Hummel, W.; Gröger, H.; Liese, A.; Herz, H.-G.;
Bornscheuer, U. T. Angew Chem 2015, 127, 2825; (b) Angew Chem
Int Ed 2015, 54, 2784.
[9] For solvent engineering on different types of Baeyer-Villiger
monooxygenases, see: (a) De Gonzalo, G.; Ottolina, G.; Zambianchi, F.;
Fraaije, M. W.; Carrea, G. J Mol Catal B: Enzym 2006, 39, 91; (b)
Secundo, F.; Fialà, S.; Fraaije, M. W.; de Gonzalo, G.; Meli, M.;
Zambianchi, F.; Ottolina, G. Biotechnol Bioeng 2011, 108, 491.
[10] Reviews on Baeyer-Villiger monooxygenases: (a) Balke, K.;
Kadow, M.; Mallin, H.; Saß, S.; Bornscheuer, U. T. Org Biomol Chem
2012, 10, 6249; (b) Mihovilovic, M. D. In Enzyme Catalysis in Organic
Synthesis 3rd ed.; Drauz, K.; Gröger, H.; May, O. Eds.; Wiley-VCH:
Weinheim, 2012; p. 1439.
Acknowledgments. The authors thank the German Federal
Environmental Foundation (Deutsche Bundesstiftung
Umwelt, DBU) for generous support within the DBU-
network
“ChemBioTec”
of
the
funding
priority
“Biotechnology” (Projects AZ 13234-32; AZ-13268-32).
[11] Drauz, K.; Gröger, H.; May, O. (Eds) Enzyme Catalysis in Or-
ganic Synthesis, 3rd ed.; Wiley-VCH: Weinheim, 2012.
[12] Sangster, J. J Phys Chem Ref Data 1989, 18, 1111.
[13] Soo, E. L.; Salleh, A. B.; Basri, M.; Rahman, R. N. Z. A.;
Kamaruddin, K. Process Biochem 2004, 39, 1511.
[14] Alfonsi, K.; Colberg, J.; Dunn, P. J.; Fevig, T.; Jennings, S.;
Johnson, T. A.; Kleine, H. P.; Knight, C.; Nagy, M. A.; Perry, D. A.;
Stefaniak, M. Green Chem 2008, 10, 31.
[15] Water solubility of isooctane and heptane according to the ma-
terial safety data sheets from Merck Millipore (download November 21,
2015): 0.56 mg/L for isooctane at 25°C; 0.05 g/L for heptane at 20°C.
[16] Bertani, G. J Bacteriol 1951, 62, 293.
[17] (a) Bisswanger, H. Practical Enzymology; Wiley-Blackwell:
Weinheim, 2011; p 5 and p. 101; (b) Cornish-Bowden, A. Fundamentals
of Enzyme Kinetics; Wiley-VCH: Weinheim, 2012; p. 85.
REFERENCES AND NOTES
[1] Weissermel, K.; Arpe, H.-J. Industrial Organic Chemistry, 4th
ed.; Wiley-VCH: Weinheim, 2003.
[2] Labet, M.; Thielemans, W. Chem Soc Rev 2009, 38, 3484.
[3] Staudt, S.; Bornscheuer, U. T.; Menyes, U.; Hummel, W.;
Gröger, H. Enzyme Microb Technol 2013, 53, 288.
[4] For the preparation of the recombinant Lactobacillus kefir al-
cohol dehydrogenase see: Weckbecker, A.; Hummel, W. Biocatal
Biotransform 2006, 24, 380.
[5] (a) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur J
Biochem 1976, 63, 175; (b) Stewart, J. D.; Reed, K. W.; Kayser, M. M.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet