A Bi-enzymatic Convergent Cascade for ε-Caprolactone Synthesis Employing 1,6-Hexanediol as a 'Double-Smart Cosubstrate'

Article abstract of DOI:10.1002/cctc.201500511

A bi-enzymatic cascade consisting of a Baeyer-Villiger monooxygenase and an alcohol dehydrogenase (ADH) was designed in a convergent fashion to utilise two molar equivalents of cyclohexanone (CHO) and one equivalent of 1,6-hexanediol as a 'double-smart cosubstrate' to produce ε-caprolactone (ECL) with water as sole by-product. The convergent enzymatic cascade reaction reported herein, is performed at ambient conditions in water, is self-sufficient with respect to cofactor, and incorporates all starting materials into the desired product, ECL. Among different enzymes explored, the reaction catalysed by cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 coupled with ADH from Thermoanaerobacter ethanolicus showed the best results, reaching 91 % conversion of CHO after 24 h with a product titre of 2 g L^-1. Scale-up of the coupled system (50 mL) performed better than the small-scale reactions and >99 % conversion of CHO and ECL concentration of 20 mM were achieved within 18 h.

Full text of DOI:10.1002/cctc.201500511

DOI: 10.1002/cctc.201500511
Communications
A Bi-enzymatic Convergent Cascade for e-Caprolactone
Synthesis Employing 1,6-Hexanediol as a ‘Double-Smart
Cosubstrate’
[a]
[b]
[c]
[a]
Amin Bornadel, Rajni Hatti-Kaul, Frank Hollmann, and Selin Kara*
A bi-enzymatic cascade consisting of a Baeyer–Villiger mono-
oxygenase and an alcohol dehydrogenase (ADH) was designed
in a convergent fashion to utilise two molar equivalents of cy-
clohexanone (CHO) and one equivalent of 1,6-hexanediol as
a ‘double-smart cosubstrate’ to produce e-caprolactone (ECL)
with water as sole by-product. The convergent enzymatic cas-
cade reaction reported herein, is performed at ambient condi-
tions in water, is self-sufficient with respect to cofactor, and in-
corporates all starting materials into the desired product, ECL.
Among different enzymes explored, the reaction catalysed by
cyclohexanone monooxygenase from Acinetobacter sp. NCIMB
Cofactor regeneration systems proposed earlier to circum-
vent the use of stoichiometric amounts of the expensive cofac-
tors used surplus amounts of the cosubstrate (e.g. isopropanol,
[
3]
ethanol) and produced stoichiometric amounts of waste.
Using 1,4-butanediol as ‘smart cosubstrate’ for the cofactor re-
generation was the next step towards more sustainable redox
[
4]
biocatalysis, which has also been validated in non-aqueous
[
5]
media.
e-Caprolactone (ECL) is a cyclic ester with a substantial
market: It is an important monomer for biodegradable, ther-
moplastic, and elastomeric polymers such as polycaprolac-
[
6]
9
871 coupled with ADH from Thermoanaerobacter ethanolicus
tone. A fully enzymatic approach for ECL synthesis in a linear
cascade fashion has been independently reported by the re-
showed the best results, reaching 91% conversion of CHO
À1
[7]
after 24 h with a product titre of 2 gL . Scale-up of the cou-
search groups of Harald Grçger and Uwe Bornscheuer, appa-
[
8]
pled system (50 mL) performed better than the small-scale re-
actions and >99% conversion of CHO and ECL concentration
of 20 mm were achieved within 18 h.
rently inspired by the studies reported in the early 90s. There-
in, oxidation of cyclohexanol (CHL) by an ADH was coupled
with further oxidation of the formed cyclohexanone (CHO) to
ECL by a BVMO. This approach is highly advantageous as it is
self-sufficient with respect to the cofactor. Another sustainable
feature that this route possesses is maximised incorporation of
the starting material that is, CHL into the final product (ECL).
More recently, extended reaction systems comprising the ADH-
BVMO linear cascade as their core have also been reported. Ef-
forts are now put on re-design and development of either up-
In recent years, biocatalytic cascades and one-pot multi-step
enzymatic reactions have attracted growing interest owing to
several important advantages: Reduction in the number of
steps required for obtaining more complex molecules coupled
with the chemo-, regio-, and stereoselectivity of various biocat-
alysts in such integrated systems potentially result in higher
[
9]
stream steps before the CHO formation or downstream steps
[1]
[10]
product yields and productivities. Redox enzymes that usually
require expensive cofactors for the electron transfer between
molecules, are nowadays mostly coupled in a cascade fash-
ion—allowing simultaneous cofactor regeneration—to perform
oxidation and reduction reactions more efficiently and more
after the ECL production in order to minimise the substrate/
product inhibition, achieve higher productivities by in situ
product removal, and finally obtain polymer precursors for ex-
[
10a]
[10b]
ample, 6-aminohexanoic acid
or oligo-ECL.
Redox-neutral cascade reactions introduced before this work
are classified into two categories: Parallel cascades (i.e. bi- sub-
strate—no intermediate—bi- or tri-product) and linear cas-
[
2]
economically.
cades (i.e. single substrate—one intermediate—single pro-
[
a] Dr. A. Bornadel, Dr. S. Kara
Institute of Microbiology
[3b,d]
duct).
The present study introduces a new class to redox-
neutral reactions, herein called a convergent cascade involving
bi-substrate and a single product without formation of an in-
termediate. We report herein on a bi-enzymatic bi-substrate
system for production of ECL, consisting of a BVMO for oxida-
tion of CHO and an ADH for oxidation of a ‘double-smart co-
substrate’ 1,6-hexanediol (1,6-HD) and simultaneous regenera-
tion of NAD(P)H (Scheme 1). On the one hand, the use of a lac-
tone-forming diol as ‘smart cosubstrate’ leads to a thermody-
namically favourable lactone coproduct, which shifts the over-
all equilibrium towards the desired product, avoiding high
surpluses of the cosubstrate and tedious product recovery, and
reducing the waste generated. On the other hand, the choice
of 1,6-HD as a ‘double-smart cosubstrate’ allows the coupling
Chair of Molecular Biotechnology
Technische Universität Dresden
0
1062 Dresden (Germany)
E-mail: selin.kara@tu-dresden.de
[
b] Prof. Dr. R. Hatti-Kaul
Department of Biotechnology
Center for Chemistry and Chemical Engineering
Lund University
P.O. Box 124, 221 00 Lund (Sweden)
[
c] Dr. F. Hollmann
Department of Biotechnology
Delft University of Technology
Julianalaan 136, 2628BL Delft (The Netherlands)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500511.
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications
Next, by making various combinations of the NADPH-depen-
dent BVMOs and ADHs, and the NADH-dependent BVMOs and
ADHs, respectively, 12 and 8 bi-enzymatic systems for the pro-
duction of ECL were designed. All 20 reactions were carried
À1
À1
out using 20 mm (2 gL ) CHO and 10 mm (1 gL ) 1,6-HD,
and the ECL formed in 48 h is shown in Figure 2. Gratifyingly,
in all of the 20 reactions CHO and 1,6-HD were converted to
ECL. The highest concentration of ECL was observed when
wild-type cyclohexanone monooxygenase (CHMO) from Acine-
tobacter sp. NCIMB 9871 was coupled with Thermoanaerobacter
ethanolicus ADH (TeSADH) (Figure 2A). Approximately 10 mm
of ECL, starting from 20 mm CHO and 10 mm of 1,6-HD, was
achieved from the preliminary trials under non-optimised con-
ditions. The second-best combination was the coupling of
ECS-Mo01 (commercial crude enzyme preparation of Acineto-
bacter calcoaceticus) with CDX-019 with ꢀ6 mm ECL formed in
Scheme 1. Synthesis of e-caprolactone (ECL) through a convergent cascade
system by coupling a Baeyer–Villiger monooxygenase (BVMO)-catalysed oxi-
dation of cyclohexanone (CHO) to ECL promoted by an alcohol dehydrogen-
ase (ADH)-catalysed oxidation of the ‘double-smart cosubstrate’ 1,6-hexane-
diol (1,6-HD) for regeneration of NAD(P)H also yielding ECL.
4
8 h. Although TADH showed the highest catalytic activity to-
wards the oxidation of 1,6-HD (Figure 1), lower oxidation activi-
ties of the NADH-dependent BVMOs (2,5-DKCMO and 3,6-
of BVMO-catalysed oxidation of CHO (two equiv) with ADH-cat-
alysed oxidation of 1,6-HD (one equiv) yielding the target com-
pound ECL (three equiv) by using only O and producing only
2
water as by-product.
In the first set of experiments we screened a range of ADHs
for activity towards 1,6-HD oxidation. Here, it is worth men-
tioning that this screening via a UV assay might only cover the
first step of the lactonisation (i.e. oxidation of the diol to the
hydroxy aldehyde). The reaction buffer system was chosen
based on a literature survey (Table SI6 in the Supporting Infor-
mation). Out of seven ADHs (3 NADPH-dependent and 4
NADH-dependent) evaluated, Thermus sp. ATN1 ADH (TADH)
showed the highest activity (Figure 1). This observation is not
surprising as TADH has been successfully applied for the oxida-
[
11]
tion of primary alcohols or a,w-diols such as 1,4-butanediol.
The activities of ADH-A and HLADH did not decrease on in-
creasing 1,6-HD concentrations (Figure SI2 (right) and SI3
(
right)). Remarkably, the commercial ADH evo 1.1.200 showed
no depletion in its activity up to 800 mm 1,6-HD (Fig-
ure SI2(left)). Among the different enzymes tested, the latter
ADH also revealed the lowest inhibition due to ECL accumula-
tion (up to 200 mm, Figure SI6). Taking all the enzymes into ac-
count, 20 mm CHO was chosen as a safe starting concentration
for our proof of concept studies.
Figure 2. Screening of BVMO and ADH couples for the synthesis of ECL pro-
moted by 1,6-HD as a ‘double-smart cosubstrate’. Reaction conditions:
+
+
c(CHO)=20 mm, c(1,6-HD)=10 mm, c(NADP /NAD )=1 mm, c(BVMO)=
À1
À1
À1
0.1 mgmL ; c(ADH)=0.01 mgmL (TeSADH, TADH, ADH-A) or 0.1 mgmL ,
buffer: Tris-HCl (100 mm, pH 8.0), 250 rpm, T=308C, 48 h. (A) Coupling of
NADPH-dependent BVMOs and ADHs, (B) Coupling of NADH-dependent
BVMOs and ADHs. Data points are average values of duplicates. Standard
deviations are <7% in Figure 2A and <5% in Figure 2B.
Figure 1. Screening of ADHs for the oxidation of 1,6-HD. Reaction condi-
+
+
tions: c(1,6-HD)=0–800 mm, c(NADP /NAD )=0.5 mm, c(ADH)=
À1
0
.1 mgmL , buffer: Tris-HCl (100 mm, pH 8.0), T=208C.
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Communications
DKCMO) towards CHO resulted in low ECL formation in those
reactions.
However, in the latter reaction, the turnover frequency (TOF)
value decreased. By increasing the substrate (i.e. CHO) concen-
tration to 100 mm (hence 1,6-HD to 50 mm), TOF stayed similar
(Reaction No 4, Table 1); however, the total conversion of CHO
was significantly reduced to 32%. A further increase in the
concentration of substrates did not improve the TOF- and con-
version values (Reaction No, Table 1), whereas conversion
values increased with additional 5-fold higher amounts of en-
zymes (Reaction No 6, Table 1).
Negative control experiments by eliminating one or two of
+
the catalytic components (NADP , CHMO, and TeSADH) were
conducted. In these control experiments, the highest ECL for-
mation (6 mm in 72 h) occurred in the absence of TeSADH (Fig-
ure SI7-C). This observation can be attributed to endogenous
ADH activity present in the crude enzyme preparation of
CHMO, utilising 1,6-HD and regenerating NADPH. Indeed, 1,6-
HD was oxidised by the CHMO preparation, however, with
a 12-fold lower activity than TeSADH (Table SI10, SI11, Fig-
ure SI8). In addition, tiny amounts of ECL (2 mm in 72 h) were
The time course of the reaction revealed the formation of
cyclohexanol (CHL) as by-product. This ‘back reduction reac-
tion’ of CHO to CHL catalysed by the ADH was also reported
+
[9b]
detected when no NADP was externally added (Figure SI7A).
by Oberleitner et al. (2014), during a linear cascade reaction.
+
This presumably results from NADPH/NADP present in the
Indeed, the kinetic analysis of CHO reduction and 1,6-HD oxi-
dation catalysed by TeSADH showed 19-fold higher Vmax for the
reduction reaction, whereas similar KM values for CHO and 1,6-
HD were observed (Table SI12). The maximum CHL concentra-
tion achieved was 7 mm after 24 h (Figure SI11, Reaction No 3),
which gradually converted to ECL through its re-oxidation to
CHO. After 72 h, no CHL was detected.
[12]
cell extract preparations (ꢀ0.2 mm), as also reported in the
[
13]
literature. No ECL formation was observed in the absence of
CHMO (Figure SI7-B and D).
Incubation of ECL in the reaction buffer (100 mm Tris-HCl,
pH 8.0) without or with the cell-free extracts of Escherichia coli
(
strain without expression plasmid) showed ca. 40% depletion
in mass (in 72 h, Figure SI9). In the screening experiments
given above (in 48 h, data not shown) ca. 20% of the total an-
alyte mass was depleted. These mass imbalance issues can to
a large extent be the result of an undesired hydrolysis of ECL.
Furthermore, evaluation of stability of enzymes without or
with their substrates (CHO, BVMOs; 1,6-HD, ADHs) exhibited re-
duction in their catalytic activities upon incubation, especially
for enzymes produced in-house (Figure SI10). This is most
probably owing to stabilizers (e.g. polyols, salts, sugars etc.)
present in commercial enzymes.
The practical usefulness of the BVMO-ADH coupled system
promoted by 1,6-HD as the ‘double-smart cosubstrate’ was
demonstrated on a semi-preparative scale (50 mL reaction
À1
À1
volume) using 98 mg (2 gL , 20 mm) CHO and 59 mg (1 gL ,
10 mm) 1,6-HD. The scaled up reaction progressed better than
the small-scale reactions and >99% conversion of CHO and
19.7 mm ECL were achieved within 18 h. This partly results
from an efficient O supply (50 mL reaction in 500 mL flask).
2
After 24 h 19 mm ECL and 3.7 mm 1,6-HD were detected (Fig-
ure SI12, SI13). Work-up of the reaction mixture gave 146 mg
1
Encouraged by the promising results obtained under non-
optimised conditions (Figure 2), we aimed for enhancing the
productivity of the CHMO-TeSADH coupled system. To this
end, higher amounts of the enzymes and substrates were used
for the reactions presented in Table 1. As shown for the Reac-
tion No 1 (the data from the CHMO-TeSADH coupled system
presented in Figure 2A), the conversion of CHO reached 71%
and 90% after 24 and 72 h, respectively. By adding another ali-
quot of CHMO after 48 h, conversion was increased to 99%
after 72 h. By using two-fold higher amounts of enzymes, full
conversion was achieved within 48 h (Reaction No 3, Table 1).
yellowish oily substance; its H NMR analysis revealed a mixture
of three compounds: ECL, poly-ECL and 1,6-HD (1:1.5:0.6) (Fig-
ure SI14). The turnover numbers (TONs) obtained were 5795,
38000, and 20 for CHMO, TeSADH and for the nicotinamide co-
factor, respectively. Through further process optimisation, for
example, establishing reactions conditions enabling the use of
nicotinamide cofactor at low amounts (<0.1 mm) and continu-
ous production, higher TONs for the nicotinamide cofactor are
achievable.
In summary, we have developed a highly atom-efficient syn-
thesis route for ECL, which is an important polymer precursor
[a]
Table 1. Conversion of cyclohexanone (CHO) and 1,6-hexanediol (1,6-HD) into e-caprolactone with internal cofactor regeneration.
[
b]
Reaction CHO 1,6-HD CHMO
number [mM] [mM] [mgmL
TeSADH
[mgmL
TOF
Conversion over time
À1
À1
À1
]
]
[min
]
5 h
24 h
48 h
c(ECL) [mM]
72 h
c(ECL) [mM]
[c]
[c]
[c]
[c]
[
%] c(ECL) [mM]
[%] c(ECL) [mM]
[%]
[%]
90.1 8.8
1
2
3
4
5
6
20
20
20
10
10
10
50
0.1
0.1
0.2
0.2
0.2
1
0.01
0.01
0.02
0.02
0.02
0.1
4.6
4.1
2.0
1.8
2.3
0.9
34.7 2.5
27.4 2.3
36.4 2.3
6.8 2.0
4.1 1.3
19.5 4.8
70.6 9.5
57.3 6.8
90.5 9.4
20.9 7.2
7.0 4.2
86.5 9.6
72.4 9.4
[
d]
e]
>99
>99
32.2 13.7
10.5 4.2
76.1 23.9
13.3
16.8
>99
18.3
26.0 11.5
7.9 4.4
67.7 34.7
[
100
200 100
100 50
51.1 25.8
+
À1
À1
[
a] Reaction conditions: c(CHO)=20:100:200 mm, c(1,6-HD)=10:50:100 mm, c(NADP )=1 mm, c(CHMO)=0.1–1 mgmL , c(TeSADH)=0.01–0.1 mgmL
,
Tris-HCl (100 mm, pH 8.0), 250 rpm (orbital shaking). Reactions (1 mL of total volume) run in 4 mL glass-vials. [b] Turnover frequency (TOF) represents mmo-
ECL formed in one minute by using mmolBVMO+ADH, determined over the first five hours (sampling at 0/1/3/5 h). [c] Based on the consumption of CHO deter-
l
mined by GC analysis. [d] The same amount of CHMO was added after 48 h. [e] The data for Reaction No 4 represent average values of duplicates, whereby
the standard deviation was <9%. Reaction temperature was 308C for the Reaction No 1 whereas 208C for the Reactions No 2–6.
ChemCatChem 2015, 7, 2442 – 2445
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Communications
with a global demand in the range of multi-kilotons. This is
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ADH-catalysed oxidation of 1,6-HD complementing the BVMO
route by regenerating the NAD(P)H and converging to the
same product ECL. The CHMO from Acinetobacter sp. NCIMB
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Acknowledgements
S.K. thanks DECHEMA e.V. (Frankfurt am Main, Germany) for fi-
nancial support in the form of a “Max Buchner” fellowship. A.B.
thanks Gesellschaft von Freunden und Fçrderern der Technische
Universität Dresden e.V. for six months of personal funding. Spe-
cial thanks to Dr. Max Steinhagen and Prof. Dr. Marion B. An-
sorge-Schumacher for fruitful discussions on molecular studies.
The authors gratefully acknowledge Dr. Jçrg H. Schrittwieser for
helpful suggestions and proofreading the manuscript. The au-
thors would like to thank (1) Prof. Dr. Wolfgang Kroutil (Universi-
ty of Graz, Austria) for the plasmids hosting alcohol dehydrogen-
ases from Rhodococcus ruber (ADH-A), Thermoanaerobacter
ethanolicus (TeSADH), and from Lactobacillus brevis (LbADH), (2)
Prof. Dr. Marco W. Fraaije (University of Groningen, The Nether-
lands) for the plasmids hosting cyclohexanone monooxygenases
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(
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Keywords: alcohol dehydrogenases · caprolactone · Baeyer–
Villiger reaction
·
monooxygenases
·
multi-enzymatic
Received: May 7, 2015
cascades · smart cosubstrate
Published online on July 14, 2015
ChemCatChem 2015, 7, 2442 – 2445
www.chemcatchem.org
2445
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