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
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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