An enzyme cascade synthesis of ε-caprolactone and its oligomers

Article abstract of DOI:10.1002/anie.201410633

Poly-ε-caprolactone (PCL) is chemically produced on an industrial scale in spite of the need for hazardous peracetic acid as an oxidation reagent. Although Baeyer-Villiger monooxygenases (BVMO) in principle enable the enzymatic synthesis of ε-caprolactone (ε-CL) directly from cyclohexanone with molecular oxygen, current systems suffer from low productivity and are subject to substrate and product inhibition. The major limitations for such a biocatalytic route to produce this bulk chemical were overcome by combining an alcohol dehydrogenase with a BVMO to enable the efficient oxidation of cyclohexanol to ε-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed ε-CL in the aqueous phase by using lipase A from Candida antarctica, thus efficiently solving the product inhibition problem and leading to the formation of oligo-ε-CL at more than 20 g L^-1 when starting from 200 mM cyclohexanol. This oligomer is easily chemically polymerized to PCL.

Full text of DOI:10.1002/anie.201410633

Angewandte
Chemie
DOI: 10.1002/anie.201410633
Biocatalysis
An Enzyme Cascade Synthesis of e-Caprolactone and its Oligomers**
Sandy Schmidt, Christian Scherkus, Jan Muschiol, Ulf Menyes, Till Winkler, Werner Hummel,
Harald Grçger, Andreas Liese, Hans-Georg Herz, and Uwe T. Bornscheuer*
Abstract: Poly-e-caprolactone (PCL) is chemically produced
on an industrial scale in spite of the need for hazardous
peracetic acid as an oxidation reagent. Although Baeyer–
Villiger monooxygenases (BVMO) in principle enable the
enzymatic synthesis of e-caprolactone (e-CL) directly from
cyclohexanone with molecular oxygen, current systems suffer
from low productivity and are subject to substrate and product
inhibition. The major limitations for such a biocatalytic route
to produce this bulk chemical were overcome by combining an
alcohol dehydrogenase with a BVMO to enable the efficient
oxidation of cyclohexanol to e-CL. Key to success was
a subsequent direct ring-opening oligomerization of in situ
formed e-CL in the aqueous phase by using lipase A from
Candida antarctica, thus efficiently solving the product inhib-
ition problem and leading to the formation of oligo-e-CL at
more than 20 glÀ1 when starting from 200 mm cyclohexanol.
This oligomer is easily chemically polymerized to PCL.
only modest selectivity (85–90%), further drawbacks arise
from the perspective of toxicity, ecology, and safety.
One obvious enzymatic alternative for the production of
e-CL is the use of Baeyer–Villiger monooxygenases
(BVMO).[4] These flavin-dependent enzymes only require
molecular oxygen as an oxidation reagent and the cofactor
NADPH. Within this enzyme class, the cyclohexanone
monooxygenase (CHMO) from Acinetobacter calcoaceticus,
which was already described almost 40 years ago,[5] is the
preferred candidate. Furthermore, this enzyme can be
produced recombinantly in yeast[6] and E. coli.[7] To date,
however, biocatalytic large-scale production of e-CL has not
been achieved for a number of reasons. These include stability
of the enzyme, the issue of cofactor regeneration, and the
need for a stoichiometric amount of a co-substrate. The major
challenge is to overcome substrate and product inhibition of
the CHMO. To enable an economic process, cheap and
efficient recycling of the cofactor NADPH without the need
for an external co-substrate is necessary. We recently reported
such a system, in which this CHMO is combined with an
alcohol dehydrogenase (ADH from Lactobacillus kefir or
a designed polyol dehydrogenase from Rhodobacter sphaer-
oides, left part of Scheme 1) to create self-sufficient cofactor
Biocatalytic processes are well established for the synthesis
of high-value fine chemicals, especially for chiral pharma-
ceutical intermediates, by using natural or engineered
enzymes.[1] In contrast, examples for the enzymatic synthesis
of bulk chemicals are still rare.[2]
e-caprolactone (e-CL, 3) is an important industrial
chemical that is currently produced at a multi-10000 ton
scale per year by the UCC process to serve as a precursor for
polymer synthesis.[3] In this process, cyclohexanone is oxidized
by using stoichiometric amounts of peracetic acid. Besides
[*] Dipl.-Biochem. S. Schmidt, Dipl.-Biochem. J. Muschiol,
Prof. Dr. U. T. Bornscheuer
Scheme 1. Synthesis of oligo-e-caprolactone (oligo-e-CL) 4 through an
enzyme cascade. First, cyclohexanol 1 is oxidized to cyclohexanone 2
by an alcohol dehydrogenase (ADH), followed by Baeyer–Villiger
oxidation by a cyclohexanone monooxygenase (CHMO) to produce
e-CL (3), with concurrent recycling of the cofactor NADPH. Severe
product inhibition is completely avoided and significantly higher
productivity is achieved through the use of lipase CAL-A as a result of
its acyltransferase activity in an aqueous system. The result is the
formation of oligo-e-CL only, without the formation of 6-hydroxycaproic
acid.
Institute of Biochemistry
Dept. of Biotechnology & Enzyme Catalysis, Greifswald University
Felix-Hausdorff-Strasse 4, 17487 Greifswald (Germany)
E-mail: uwe.bornscheuer@uni-greifswald.de
Dr. U. Menyes
Enzymicals AG, Walther-Rathenau-Strasse 49a
17489 Greifswald (Germany)
M.Sc. T. Winkler, Prof. Dr. W. Hummel, Prof. Dr. H. Grçger
Organic Chemistry I, Faculty of Chemistry, Bielefeld University
P.O. Box 100131, 33501 Bielefeld (Germany)
Dipl.-Biochem. C. Scherkus, Prof. Dr. A. Liese
Institute of Technical Biocatalysis
Hamburg University of Technology TUHH
Denickestrasse 15, 21073 Hamburg (Germany)
recycling when starting from the readily available bulk
chemical cyclohexanol.[10] These studies revealed that even
at concentrations of 60 mm, severe product inhibition by e-CL
takes place in addition to modest inhibition by cyclohexanol
1 and cyclohexanone 2.[8]
Dr. H.-G. Herz
Polymaterials AG, Innovapark 20, 87600 Kaufbeuren (Germany)
Substrate inhibition can easily be addressed through
appropriate feeding of cyclohexanol. When both ADH and
CHMO are expressed at sufficiently high levels, the concen-
tration of cyclohexanone as an in situ formed intermediate
also remains at low levels. By contrast, product inhibition and
[**] We thank the “Deutsche Bundesstiftung Umwelt” for financial
support (AZ 13268-32).
Supporting information for this article (including experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201410633.
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Angewandte
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enzyme deactivation by the product e-CL, particularly at
higher concentrations, represents a major hurdle to overcome.
Several routes to remove this fully water-miscible lactone by
in situ extraction with an organic solvent or the use of
adsorbents suffer substantially from low productivity and
additional costs and environmental disadvantages.
Herein, we report an elegant solution in which the e-CL
produced by the one-pot two-step enzymatic method is
directly subjected to in situ ring-opening oligomerization by
using lipase CAL-A from Candida antarctica[9] (Scheme 1).
Such a process solves the problem of enzyme inhibition and
deactivation by e-CL (3) at higher concentrations. Conven-
iently, the formed oligo-e-CL is hydrophobic and can be
isolated by extraction or precipitation. Although the enzy-
matic synthesis of polymers, especially enzymatic ring-open-
ing polymerization, is well documented,[10] the formation of
polyesters always required bulk organic solvents and notably
the absence of water to prevent undesired hydrolysis.[11]
Interestingly, lipase CAL-A has unique acyltransferase activ-
ity that enables efficient formation of the ester product
despite the presence of bulk amounts of water.[12] Indeed,
when we determined the hydrolytic activity of CAL-A
towards 1m of 3 in aqueous solution, we unexpectedly did
not detect any hydrolysis of this ester. After a few hours of
incubation, we observed the formation of a white precipitate
in the aqueous phase (Figures S1,S2 in the Supporting
Information), which after isolation and examination by gel
permeation chromatography revealed that CAL-A catalysis
led to oligomers of compound 3 with a maximum molecular
weight of 1200 gmolÀ1 (Table 1, peak retention volume
16.4 mL; Figure 1). Both end groups can be specified from
Figure 1. GPC chromatogram of oligo-e-CL 4 synthesized directly by
CAL-A (after extraction from the biocatalysis reaction). The single
narrow peak represents the internal standard toluene; the various
peaks between retention volumes 14.80 to 20.30 mL correspond to
oligomer 4.
and studied the effect of CAL-A in one-pot biocatalysis
reactions at different cyclohexanol concentrations. The con-
centration of 3 strongly decreased in the presence of CAL-A
compared to the biotransformation without the addition of
CAL-A (Figure 2). The lower concentration of e-CL presum-
Table 1: Characterization of oligo- and poly-e-caprolactone obtained
through different routes and analyzed by GPC.
Product[a]
Peak retention
volume [mL]
Mn [Da]
Mw [Da]
Mw/Mn
PCLchem
12.0
18.4
16.4
12.2
35.426
160
615
48.248
375
1154
1.4
2.3
1.8
1.7
Oligo-e-CLCascade
Oligo-e-CLCAL-A
Oligo-e-CLCond.
21.662
37.773
[a] PCLchem: chemically synthesized; Oligo-e-CLCascade: obtained by using
all three enzymes starting from 1; Oligo-e-CLCAL-A : obtained by using
lipase A starting from 1m 3; Oligo-e-CLCond: chemically polymerized
Figure 2. Comparison of the e-CL concentration in biocatalysis by ADH
and wild-type CHMO with (grey bars) and without (black bars) the
addition of CAL-A. CAL-A leads to substantially lower levels of e-CL as
a result of the conversion of e-CL into oligo-e-CL.
Oligo-e-CLCAL-A
.
the expected enzymatic reaction mechanism, which is iden-
tical to the ring-opening polymerization performed in organic
solvents.[10h] These end groups are thus a carboxyl moiety and
an alcohol moiety.
This oligomer (oligo-e-CLCAL-A, Table 1) could be easily
converted chemically into high-molecular-weight polymer
(oligo-e-CLCond.), which showed properties identical to stan-
dard chemically synthesized poly-e-caprolactone (PCLchem).
1H NMR spectroscopy was used to confirm the identity of the
two polymers (Figure S3–S5).
ably results from the CAL-A-catalyzed formation of oligo-e-
CL. With 1 glÀ1 (per batch) of lyophilized CAL-A during the
biotransformation, much lower concentrations of e-CL were
observed compared to reactions without CAL-A, in which
only e-CL was formed. This result strongly indicates the
conversion of e-CL into oligo-e-CL, and the conversion was
confirmed by 1H NMR spectroscopy analysis (Figure S5).
Furthermore, these experiments demonstrated that whole-
cell biotransformation could be performed with all three
enzymes (ADH, CHMO and CAL-A) in a single one-pot
cascade reaction.
Next, we used ADH and CHMO as a mixture of two E.
coli cell suspensions containing these recombinant enzymes
2
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In addition to inhibition of the CHMO by 3, the instability
of this enzyme is a further reason why large-scale enzymatic
production of 3 has not so far been achieved. To overcome
this problem, the double mutant C376L/M400I of CHMO was
used since this double mutation has been reported[13] to confer
higher oxidative and long-term stability on CHMO.
Preliminary experiments showed that expressing ADH
and BVMO in separate E. coli cells is superior to expressing
them in a single whole-cell system. Different ratios of whole-
cell cultures containing recombinantly expressed ADH or the
BVMO mutant C376L/M400I were thus used together for the
biotransformation of cyclohexanol to e-CL (Figure S7). The
optimal ratio of ADH and CHMO is a very important factor
because of differences in expression levels, specific activity,
and stability between the two enzymes. At 60 mm 1, the
highest conversion was achieved at an ADH/CHMO ratio of
1:10.
Although the system is in principle self-sufficient with
respect to cofactor recycling (Figure S6), we observed that the
addition of acetone and glucose is useful for faster regener-
ation of NADPH at high substrate loading (Figure S8) since
the two enzymes are expressed in separate E. coli cells
(Figure S6). A stoichiometric amount of acetone gave a con-
version of 95%.
intermediate for the production of monomeric 6-aminohex-
anoic acid.[14] This further underlines the power of BVMOs
for organic synthesis and especially the importance of enzyme
cascade synthesis of e-CL as a starting point to address
different fields of application.
Received: October 31, 2014
Revised: December 3, 2014
Published online: && &&, &&&&
Keywords: Baeyer–Villiger monooxygenases ·
.
cascade reactions · e-caprolactone · enzyme catalysis ·
polymer synthesis
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reaction (Scheme 1).
1 (mm)
ADH
CHMO À1
CAL-A
[kU][a]
Conversion
[%][b]
À1
[mUmgcells
]
[mUmgcells ]
200
300
500
1.78Æ0.20
1.89Æ0.17
1.93Æ0.23
3.32Æ0.18
3.56Æ0.10
3.45Æ0.20
30
30
30
99.0Æ0.7
74.0Æ1.3
43.0Æ2.1
[a] Determined against the standard substrate tributyrin. [b] Based on
consumption of 1 as determined by GC analysis.
of substrate with the same amounts of the enzymes, 74% and
43% conversion, respectively, were observed. Analysis of the
product from the 200 mm reaction by GC analysis and
1H NMR spectroscopy confirmed that 75% oligomer was
formed with 25% e-CL present as co-product, which obvi-
ously can be easily co-extracted with the oligomer.
In summary, we disclose a biocatalytic approach to the
production of e-caprolactone followed by direct in situ trans-
formation into oligo-e-caprolactone. This method leads to
high product yields at substrate concentrations of more than
200 mm. This elegant setup enables the combination of
monomer formation with direct in situ oligomer synthesis at
substrate concentrations exceeding those of previous studies
by at least six-fold. Future work will address further improve-
ment of the overall productivity by using bioprocess engineer-
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recently reported a related cascade process with e-CL as an
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Communications
Biocatalysis
S. Schmidt, C. Scherkus, J. Muschiol,
U. Menyes, T. Winkler, W. Hummel,
H. Grçger, A. Liese, H.-G. Herz,
U. T. Bornscheuer*
&&& — &&&
An Enzyme Cascade Synthesis of
e-Caprolactone and its Oligomers
Let’s polymerize! Oligo-e-caprolactone
was produced in a one-pot enzymatic
cascade synthesis starting from cyclo-
hexanol. In the first step, cyclohexanol is
oxidized by an alcohol dehydrogenase
(ADH) in combination with the cyclo-
hexanone monooxygenase (CHMO) from
Acinetobacter calcoaceticus, followed by
direct ring-opening oligomerization of e-
caprolactone in an exclusively aqueous
phase by lipase A from Candida antarctica
(CAL-A).
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