Rapid conversion of cyclohexenone, cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum candidum CCT 1205

Article abstract of DOI:10.1080/10242422.2017.1310207

?-Caprolactone (?-CL) was obtained with excellent conversion and short reaction times from the substrates cyclohexenone, cyclohexanone and cyclohexanol using whole cells of Brazilian Geotrichum candidum (CCT 1205). The reactions were monitored over time by gas chromatography, and the intermediates of the one-pot cascade biotransformation involving reductions of C=C and C=O bonds as well as the Baeyer–Villiger oxidation were identified and quantified. The Baeyer–Villiger monooxygenase (BVMO) enzyme was predominant, and all three substrates were completely converted into ?-CL. Furthermore, the whole cells of Geotrichum candidum were recycled and reutilized in the biotransformation of cyclohexanone, producing ?-CL at least six consecutive times without a significant loss of activity, reaction yields or product purity.

Full text of DOI:10.1080/10242422.2017.1310207

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Rapid conversion of cyclohexenone,
cyclohexanone and cyclohexanol to ε-caprolactone
by whole cells of Geotrichum candidum CCT 1205
André L. Patrício Silva, Poliane K. Batista, Augusto D. Filho, Claudionor S. do
Nascimento Junior, Julio S. Rebouças & Juliana A. Vale
To cite this article: André L. Patrício Silva, Poliane K. Batista, Augusto D. Filho, Claudionor
S. do Nascimento Junior, Julio S. Rebouças & Juliana A. Vale (2017): Rapid conversion of
cyclohexenone, cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum
candidum CCT 1205, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2017.1310207
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1310207
Published online: 18 Apr 2017.
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Date: 02 May 2017, At: 06:07

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
http://dx.doi.org/10.1080/10242422.2017.1310207
RESEARCH ARTICLE
Rapid conversion of cyclohexenone, cyclohexanone and cyclohexanol to
e-caprolactone by whole cells of Geotrichum candidum CCT 1205
Andr ꢀe L. Patr ꢀı cio Silva, Poliane K. Batista, Augusto D. Filho, Claudionor S. do Nascimento Junior,
Julio S. Rebou c¸ as and Juliana A. Vale
Departamento de Qu ꢀı mica, Universidade Federal da Para ꢀı ba, Jo ~a o Pessoa, Brazil
ABSTRACT
ARTICLE HISTORY
e-Caprolactone (e-CL) was obtained with excellent conversion and short reaction times from the
substrates cyclohexenone, cyclohexanone and cyclohexanol using whole cells of Brazilian
Geotrichum candidum (CCT 1205). The reactions were monitored over time by gas chromatog-
raphy, and the intermediates of the one-pot cascade biotransformation involving reductions of
C¼C and C¼O bonds as well as the Baeyer–Villiger oxidation were identified and quantified. The
Baeyer–Villiger monooxygenase (BVMO) enzyme was predominant, and all three substrates were
completely converted into e-CL. Furthermore, the whole cells of Geotrichum candidum were
recycled and reutilized in the biotransformation of cyclohexanone, producing e-CL at least six
consecutive times without a significant loss of activity, reaction yields or product purity.
Received 16 August 2016
Revised 26 December 2016
Accepted 16 February 2017
KEYWORDS
Biotransformation;
Baeyer–Villiger monooxyge-
nase; e-caprolactone;
Geotrichum candidum and
cascade reaction
Introduction
Carballeira et al. (2002) achieved good results in a
2-hour reaction by applying growing and immobi-
7
The oxidation of ketones through the Baeyer–Villiger
reaction is of extreme importance in chemical synthe-
sis (Punniyamurthy et al. 2005) and industrial proc-
esses (Leisch et al. 2011). For example, the industrial
oxidation of cyclohexanone produces the e-caprolac-
tone (e-CL) monomer, which is used in the manufac-
ture of poly-caprolactone, a polymer that is used in
the manufacture of adhesives, dyes and compounds
used for environmental protection and medical pur-
poses due to its biocompatibility, non-toxicity and bio-
degradability (Weissermel and Arpe 2003).
lized cells of Geotrichum candidum NCYC49 for the
production of e-CL from cyclohexanone. In a similar
experiment, Mandal et al. (2002) applied whole cells of
Fusarium sp. and obtained quantitative yields of e-CL
(30% wet weight) after 48 hours of reaction.
Starting from cyclohexanol as a substrate, e-CL is
obtained by two combined biocatalytic oxidation
steps. The first step involves the catalytic alcohol
dehydrogenase (ADH) enzyme and provides an inter-
þ
mediary cyclohexanone, while the NADP cofactor
(oxidized form) is converted to NADPH (reduced form).
The high interest in this reaction is due to its high
chemo-, stereo- and enantioselectivity control (Brink
et al. 2004). The most studied chemical method for
the oxidation of ketones uses peracids, particularly m-
chloroperbenzoic acid (Hussain et al. 2014; Yachnin
et al. 2014); however, a search for safer, more selective
and environmentally friendly methods is underway.
Biotransformation has emerged as an alternative and
efficient method for the synthesis of lactones and
esters by transformation from different substrates by
the Baeyer–Villiger monooxygenases (BVMOs), which
are found mainly in several microorganisms (Ratuꢀs
et al. 2009). BVMOs are known as flavoenzymes and
require NADPH for their activity (Mihovilovic et al.
In a second oxidation reaction, cyclohexanone is then
transformed in situ to yield e-CL by nucleophilic oxy-
genation in the presence of BVMO and NADPH (Mallin
et al. 2013).
Staudt et al. (2013), using an ADH from
Lactobacillus kefir and BVMO from Acinetobacter cal-
coaceticus, with both enzymes recombinantly overex-
pressed in E. coli, showed that the desired product,
e-CL, was obtained with greater than 94% conversion
when operating at a cyclohexanol concentration in the
range of 20–60 mM.
With enone substrates, reduction of the C¼C dou-
ble bond catalysed by enoate reductase in the pres-
ence of reduced nicotinamide cofactor NADPH was
2006; Cuetos et al. 2012).
CONTACT Juliana A. Vale
julianadqf@yahoo.com.br
Departamento de Qu ꢀı mica, Universidade Federal da Para ꢀı ba, Jo ~a o Pessoa, PB 58051-970, Brazil
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
A. L. P. SILVA ET AL.
observed (Paul et al. 2013). Using recombinant cells in
the Baeyer–Villiger biooxidation, it was observed that
the bioreduction reaction of the double bond of
enones occurs prior to oxidation to the corresponding
lactone (Mihovilovic et al. 2006).
washed twice with distilled water to remove residual
culture medium.
Reaction methodologies
The washed Geotrichum candidum biomass was
weighed and placed into an Erlenmeyer flask (125 mL)
containing 10 mL of phosphate buffer (pH 6.5), and
In this study, the self-sufficient Baeyer–Villiger bio-
transformation of three substrates was evaluated
(
cyclohexenone, cyclohexanone and cyclohexanol) for
1
0 lL (ꢃ100 lmol) of the described substrates was
the synthesis of e-CL using whole cells of Geotrichum
candidum from the Brazilian tropical culture collection
ꢁ
added. The flasks were incubated at 28 C and
180 rpm in an orbital shaker. The aliquots of the reac-
(
CCT 1205) as biocatalysts. The advantage of using
tion mixture were extracted with ethyl acetate, and
the organic phases were analysed using gas chroma-
tography. Recycling and reuse tests of the Geotrichum
candidum cells were conducted by filtering the cells
and washing them three times with distilled water
after each reaction. All experiments were performed in
triplicate.
whole cells is that they make the process less complex
than the use of genetically modified biocatalysts, while
eliminating the use of synthetic cofactors, which are
very expensive. Cascade biotransformations via oxida-
tion and reduction processes were also evaluated by
monitoring the time courses on each of the substrates.
Materials and methods
Results and discussion
Chemicals
Preliminarily, the biotransformation of cyclohexenone
(
1) was studied to evaluate the cascade reaction
Cyclohexenone, cyclohexanone, cyclohexanol and e-CL
were obtained from Sigma-Aldrich (St. Louis, MO).
Glucose, malt extract, yeast extract and peptone were
obtained from HIMEDIA (Mumbai, India), and ethyl
acetate and other solvents were obtained from TEDIA
through one-pot reduction and oxidation processes. In
the initial reactions, two culture media (YM and
Sabouraud) were tested based on the wet weight of
the cells of Geotrichum candidum CCT 1205.
The reactions only achieved substrate conversion to
e-CL in YM culture media. The presence of glucose
inhibits oxidation reactions, and only reduction prod-
ucts of the C¼C and C¼O bonds are obtained. To
optimize the results, several tests were carried out
with variations of the whole-cell concentrations of
Geotrichum candidum and the substrate concentration.
The reaction progress was monitored using gas chro-
matography with commercial standards, and the
results are shown in Table 1.
(
Fair Lawn, NJ).
Analytical
Chromatographic analyses were performed with a GC-
QP2010-Shimadzu chromatograph equipped with an
FID detector using an Rtx-5 fused capillary column
(
30 m ꢀ 0.25 mm ꢀ0.25 mm film thickness) and N as a
2
carrier gas (1 mL/min). The used heating gradient was
6
and 240 C (45 C/min).
ꢁ ꢁ ꢁ ꢁ ꢁ
0 C (1 min), 100 C (35 C/min), 150 C (25 C/min),
As seen in Table 1, the complete biotransformation
of cyclohexenone (1) is slow at high substrate concen-
trations (entries 2 and 3), and only ene-reductase
ꢁ
ꢁ
(ERED) and ADH enzymatic activities were observed,
Microorganisms and culture conditions
which predominantly indicates intermediates 2 and 3.
In contrast, when the proportion of whole cells was
increased (entry 4), BVMO was significantly more
active, and substrate 1 was completely converted to
e-CL (4).
The reaction time of 24 hours to evaluate the con-
centration of product 4 was chosen arbitrarily and is
significantly longer than the time required to convert
1 to 4. To better understand the catalytic activity of
the cascade reaction and to evaluate the concentra-
tions of the intermediates as a function of time during
the biotransformation, a time course of the reaction
Geotrichum candidum CCT 1205, which was used in
this study, was acquired from the CCT collection (CCT-
Cole c¸ ~a o de Cultura Tropical de Pesquisas, Funda c¸ ~a o
Andr ꢀe Toselo, Campinas, Brazil) and grown in an
Erlenmeyer flask (500 mL) containing 250 mL of auto-
ꢂ1
claved yeast extract medium YM (D-glucose 10.0 g L ),
ꢂ1
ꢂ1
malt extract (3.0 g L ), yeast extract (3.0 g L ) and
ꢂ1
peptone (5.0 g L ). The inoculum was grown under
orbital shaking (200 rpm) for 48 hours. The Geotrichum
candidum cells were separated from the broth by cen-
trifugation (2500 rpm) for 10 min, and the biomass was

BIOCATALYSIS AND BIOTRANSFORMATION
3
Table 1. Biotransformation of cyclohexenone under various conditions.
a
b
Entry
Biomass (g)
Substrate (mM)
1 (%) cyclohexenone
2 (%) cyclohexanone
3 (%) cyclohexanol
4 (%) e-CL
1
2
3
4
a
1.0
1.0
2.0
3.0
3.1
10.3
10.3
10.3
0
63
29
0
67
33
62
0
14
4
9
18
0
0
0
100
The conversion percentage was determined using gas chromatography.
b
Biomass refers to the wet weight of the cells.
Figure 1. Reaction time course of the biotransformation of cyclohexenone. Conditions: 3 g of whole cell biomass, 10 lL of sub-
strate, 10 mL of pH 6.5 phosphate buffer, six hour reaction time, and agitation at 180 rpm. Conversion of intermediates and prod-
uct was monitored by gas chromatography.
using entry 4, but with a last reaction time of six
hours, was determined using gas chromatography to
monitor aliquots extracted from the reaction medium
every hour (Figure 1).
As seen in Figure 1, first, the C¼C enone endocyclic
bond was reduced by ERED enzyme, and then the
C¼O bond was reduced by ADH enzyme (Scheme 1).
After two hours of reaction, substrate 1 was com-
pletely consumed, and e-CL (4) concentrations
increased due to the subsequent oxidation of inter-
mediates 2 and 3 by ADH and BVMO, respectively.
Throughout the reaction, the concentration of product
the desired product e-CL (4) was obtained, with quan-
titative yield. The kinetic profile shows that the conver-
sion of 1 to 4 was extremely fast, requiring only five
hours of reaction time. Our results corroborate the
results of Carballeira et al. (2002), which showed that
the production of caprolactone involves an equilibrium
between intermediates 2 and 3. The yields obtained
using the one-pot cascade biotransformation described
here are much higher than those obtained by
Mihovilovic et al. (2006), who obtained only 52% e-CL
conversion in 48 hours using BVMO expressed in
E. coli.
4
increased as the concentration of intermediates 2
To better understand the equilibrium between
intermediates 2 and 3 during the conversion of e-CL,
and 3 decreased, and after five hours of reaction, only

4
A. L. P. SILVA ET AL.
the biotransformation from cyclohexanone (2) and
cyclohexanol (3) as substrates using Geotrichum candi-
dum cells was studied. First, the best conditions for
the biotransformation of 2 to 4 were determined. This
reaction occurred extremely quickly when only 1 g of
whole cell biomass and 10 lL of the substrate were
used, with a quantitative yield of e-CL in just four
hours of reaction time. These results were much better
than those reported by Carballeira et al. (2002), who
obtained e-CL (4) with quantitative yield in 72 hours
using a strain of immobilized Geotrichum candidum
NCYC49 in various matrices. The kinetic profile of this
biotransformation was determined using gas chroma-
tography by monitoring aliquots extracted from the
reaction medium every 30 min (Figure 2).
As seen in Figure 2, intermediate 3 was observed
within 30 min by ADH activity, and after only one
hour, product 4 was observed by BVMO activity.
Similar to Carballeira et al. (2002), an equilibrium was
observed between substrate 2 and intermediate 3 dur-
ing the first hours of the biotransformation reaction.
Oxidation occurred simultaneously through ADH and
BVMO enzymatic activity in the reaction medium
(Scheme 2). e-Caprolactone (4) was formed rapidly in
quantitative yield within four hours.
In the chemical industry, e-CL is synthesized via the
oxidation of cyclohexanone; however, recent studies
have shown that e-CL can also be produced via the
bio-oxidation of cyclohexanol. For example, Mallin
et al. (2013) used PDH and CHMO, which are
expressed in E. coli.
The biotransformation of cyclohexanol to e-CL using
whole cells of Geotrichum candidum (CCT 1205) is
extremely rapid, and only 1 g of biomass and 10 lL of
substrate are needed for the exclusive formation of
e-CL in just four hours. The course of this reaction was
also determined and is shown in Figure 3.
Scheme 1. Microbial oxidation and reduction of cyclohexe-
none. The product of the Baeyer–Villiger oxidation is
caprolactone.
Scheme 2. Microbial oxidation and reduction of cyclohexa-
none. The product of the Baeyer–Villiger oxidation is
caprolactone.
Figure 2. Reaction time course of the biotransformation of cyclohexanone. Conditions: 1 g of whole cell biomass, 10lL of sub-
strate, 10 mL of pH 6.5 phosphate buffer, four hour reaction time, and agitation at 180 rpm. Conversion of intermediates and prod-
uct was monitored by gas chromatography.

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 3. Reaction time course of the biotransformation of cyclohexanol. Conditions: 1 g of whole cell biomass, 10 lL of substrate,
0 mL of pH 6.5 phosphate buffer, four hour reaction time, and agitation at 180 rpm. Conversion was determined using gas
1
chromatography.
recycled and reused for the biotransformation of sub-
strate 2. After simply washing the cells with buffer, six
cycles of reuse were repeated. e-Caprolactone (4) was
obtained at quantitative yield in the four first cycles, at
9
6% yield in the fifth cycle, and at 88% in the sixth
Scheme 3. Microbial oxidation of cyclohexanol. The product of
the Baeyer–Villiger oxidation is caprolactone.
cycle. Thus, although a slight decrease in enzymatic
conversion occurs, the catalytic process remains very
active even after six cycles of reusing the free cells.
As seen in Figure 3, the ADH enzyme activity
occurs rapidly, forming intermediate 2. The conver-
sion of intermediate 2 is slow, and an equilibrium
between substrate 3 and intermediate 2 is also
observed here; this is the rate-limiting step of the
overall reaction. After two hours, a second oxidation
due to the catalytic activity of BVMO results in the
appearance of e-CL (Scheme 3). The formation of
e-CL (4) occurs rapidly and in quantitative yield
within 4 hours (Figure 3).
Conclusions
The use of whole Geotrichum candidum cells obtained
from the Brazilian tropical culture collection (CCT
1205) as biocatalysts efficiently biotransformed the
substrates cyclohexenone (1), cyclohexanone (2) and
cyclohexanol (3) into e-CL (4) in quantitative yields
over short reaction times. In the biotransformation cas-
cade reaction of cyclohexanone, ERED activity catalys-
ing the first step was observed, followed by ADH and
BVMO catalysing the next steps. In the bio-oxidation
of cyclohexanone (2), an equilibrium catalysed by ADH
was clearly observed between species 2 and 3, fol-
lowed by BVMO enzymatic catalysis generating e-CL in
quantitative yield in just four hours. Similarly, when
using cyclohexanol (3) as a substrate, an equilibrium
between species 2 and 3 catalysed by ADH was also
observed, again followed by BVMO enzymatic activity,
providing similar results. These results represent the
best results to date in terms of experimental simplicity,
reaction time and e-CL (4) yields obtained using whole
cells.
The values obtained for the limit of detection (LOD)
and limit of quantification (LOQ) were determined
from the parameters of the calibration curve of capro-
lactone using GC-FID according to the equations LOD
5
3SD/b and LOQ 510SD/b, where SD is the standard
deviation of the intercept and b is the slope of the
regression line. The results (LOD ¼0.39) and (LOQ
¼
1.17) showed the method to be sensitive at the low
concentrations in all experiments performed in this
work.
Due to the rapid conversion of the three substrates
into e-CL (4), the Geotrichum candidum cells could be

6
A. L. P. SILVA ET AL.
Disclosure statement
e-caprolactone from cyclohexanol. Enzyme Microb Technol
3:283–287.
Mandal D, Ahmad A, Khan MI, Kumar R. 2002. Biocatalytic
transformation of cyclohexanone by Fusarium sp. J Mol
Catal A: Chem 181:237–241.
5
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
Mihovilovic MD, Snajdrova R, Gr o€ tzl B. 2006. Microbial
Baeyer–Villiger oxidation of 4, 4-disubstituted cyclohexan-
and cyclohexenones by recombinant whole-cells express-
ing monooxygenases of bacterial origin. J Mol Catal B:
Enzym 39:135–140.
Funding
The authors thank CAPES and CNPq for financial support.
Paul CE, Gargiulo S, Opperman DJ, Lavandera I, Gotor-
Fern ꢀa ndez V, Gotor V, Taglieber A, Arends IWCE, Hollmann
F. 2013. Mimicking nature: synthetic nicotinamide cofac-
tors for C ¼ C bioreduction using enoate reductases. Org
Lett 15:180–183.
Punniyamurthy T, Velusamy S, Iqbal J. 2005. Recent advances
in transition metal catalyzed oxidation of organic sub-
strates with molecular oxygen. Chem Rev 105:2329–2364.
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