New fungi for whole-cell biotransformation of carvone enantiomers. Novel p-menthane-2,8,9-triols production

Article abstract of DOI:10.1016/j.apcata.2013.08.034

The microbial biotransformation of carvone enantiomers by Lasiodiplodia theobromae, Trichoderma harzianum and Mucor circinelloides was investigated. Biotransformation experiments were conducted using growing or resting fungi cells and the products were an

Full text of DOI:10.1016/j.apcata.2013.08.034

Applied Catalysis A: General 468 (2013) 88–94
Contents lists available at ScienceDirect
Applied Catalysis A: General
journal homepage: www.elsevier.com/locate/apcata
New fungi for whole-cell biotransformation of carvone enantiomers.
Novel p-menthane-2,8,9-triols production
Fátima M. Nunesa, Gabriel F. dos Santosb, Natália N. Saraivaa, Marília A. Trappb,
Marcos C. de Mattosa, Maria da Conceic¸ ão F. Oliveiraa,∗, Edson Rodrigues-Filhob,∗∗
a Departamento de Química Orgânica e Inorgânica, Laboratório de Biotecnologia e Síntese Orgânica (LABS), Universidade Federal do Ceará, Campus do Pici,
Caixa Postal 6044, 60455-970, Fortaleza, CE, Brazil
b Departamento de Química, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905, São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 April 2013
Received in revised form 13 August 2013
Accepted 19 August 2013
Available online 28 August 2013
The microbial biotransformation of carvone enantiomers by Lasiodiplodia theobromae, Trichoderma
harzianum and Mucor circinelloides was investigated. Biotransformation experiments were conducted
using growing or resting fungi cells and the products were analyzed by GC–MS or LC–MS. The isolated
compounds were identified by NMR. (S)-Carvone yielded only the enone reduction product dihydrocar-
vone by M. circinelloides, while (R)-carvone yielded p-menthane-2,8,9-triols when biotransformed by L.
theobromae and M. circinelloides. These p-menthanetriols are being reported for the first time as biotrans-
formation products. Neodihydrocarveol was the only product from the biotransformation of (R)-carvone
by T. harzianum. These biotransformations can find practical applications, especially in the case of the
dihydrocarveol production.
Keywords:
Carvone
p-Menthane-2,8,9-triol
Lasiodiplodia theobromae
Trichoderma harzianum
Mucor circinelloides
Biotransformation
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
biotransformation reaction varied according to the investigated
organism. Although no product from the dihydroxylation of the
The p-menthane-2,8,9-triols 1 (Fig. 1) are monoterpenes that
were first isolated from the fruiting body of the basideomycete
Flammulina velutipes [1] and later from the water-soluble portion
of the methanol extract from the caraway fruit [2]. The absolute
configurations from six of the sixteen isomers represented by the
planar formula 1 were established [1,2], while four compounds had
only their relative configuration elucidated [2].
exocyclic double bond of carvone have been reported in the litera-
ture, this reaction was observed in the microbial biotransformation
of limonene [3].
In the course of our ongoing research on the identification
of new sources of enzymes based on the Brazilian biodiversity,
we investigated the biotransformation of (R)-(−)- and (S)-(+)-
carvone (2) by the filamentous fungi Lasiodiplodia theobromae,
Trichoderma harzianum and Mucor circinelloides. Herein, we report
for the first time compounds (1R,2S,4R,8S)-p-menthane-2,8,9-triol
(1a) and (1R,2S,4R,8R)-p-menthane-2,8,9-triol (1b) as biotransfor-
mation products.
The structures of these triols suggest that these compounds are
derived from carvone (2) by reduction of the conjugated C C and
C
O bonds and dihydroxylation of the exocyclic double bond. The
transformations of both (R)-(−)- and (S)-(+)-carvone (2) by cells
of microorganisms [3–9], plants [10–14] and microalgae [15–18]
have been described in the literature, and the products (3–9) are
displayed in Fig. 1. These products are formed by reduction of the
endocyclic C C bond (3), carbonyl group (4) or both the C C and
2. Experimental
C
O bonds (5 and 6). The products from carvone oxidation (6–9)
2.1. Microorganisms
have also been reported [3,19]. The stereochemical course of the
L. theobromae (strain BRF118) was obtained from EMBRAPA
Agroindústria Tropical (Fortaleza, CE, Brazil) where the microor-
ganism was isolated in the Laboratory of Phytopathology from
infected guava. M. circinelloides (strain LaBioMMi #009) was iso-
lated in the LaBioMMi (DQ/UFSCar, São Carlos, SP, Brazil) from the
wood of Pinus taeda collected on the UFSCar campus. T. harzianum
∗
Corresponding author. Tel.: +55 8533669144; fax: +55 8533669782.
Corresponding author. Tel.: +55 1633518053; fax: +55 1633518350.
E-mail addresses: mcfo@ufc.br, mcfo67@gmail.com (M.d.C.F. Oliveira),
∗∗
edinho@pq.cnpq.br (E. Rodrigues-Filho).
0926-860X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apcata.2013.08.034

F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
89
CH3
CH3
OH
O
H3C
CH2
H3C
CH2
4
3
CH3
CH3
OH
OH
CH3
O
OH
OH
H3C
CH2
H3C
1
5
R or S
H3C
CH2
CH3
2
CH3
O
OH
O
CH3
H3C
CH3
HO
O
CH2
O
OH
CH2
9
6
CO2H
H3C
CH2
8
H3C
CH2
7
Fig. 1. Biotransformation products of carvone (2) reported in the literature (compounds 3–9) [3,4,19] and in the present work (compounds 1, 3, 5 and 6).
(strain BRF117) was isolated in the Laboratório de Micologia e
Patologia de Sementes (Fortaleza, CE, Brazil) from cattle dung.
added at 0 ◦C, and the reaction was stirred at room temper-
ature for 3 h. The reaction was quenched by the addition of
H2O, and the products were extracted with EtOAc (3 × 10 mL).
The organic layer was dried over anhydride Na2SO4 and fil-
tered. After solvent distillation under reduced pressure, the crude
products (0.92 mg) were coarsely purified on a silica gel col-
umn (silica gel 60, 230–240 mesh; EtOAc/hexane as eluent) and
analyzed by gas chromatography–mass spectrometry (GC–MS)
recorded on a Shimadzu GCMS-QP5050A apparatus (Column OV5
[30 m × 0.25 mm × 0.25 ␮m]). A mixture of carveol (4) and dihydro-
carveol (5) was identified as the product of the chemical reduction
of (R)-carvone (2).
2.2. Chemical materials
(R)- and (S)-Carvone (2) were purchased from Sigma–Aldrich®.
All of the other chemicals (NaBH4, anhydride Na2SO4, glucose,
NaNO3, KH2PO4, MgSO4, FeSO4·7H2O, MgSO4, KCl and NaCl)
were obtained from Sigma–Aldrich® or Fluka®. Potato dextrose
agar (PDA) and potato dextrose broth (PDB) were obtained from
Acumedia®. Column chromatography was performed on silica gel
60 (230–240 mesh) from Vetec®. Solvents were purchased from
Qhemis®, Sinth® or Vetec®, and HPLC grade solvents were acquired
from Mallinckodt® and Tedia®. Analytical TLC analyses were per-
formed on aluminum sheets pre-coated with silica gel 60 F254
(0.2 mm thick) from Merck®.
2.4. Carvone biotransformation by growing cells of L. theobromae
2.4.1. Minimum inhibitory concentration (MIC) of (R)-carvone on
L. theobromae growth
2.3. Chemical reduction of (R)-carvone
Two 3 mm disks of medium containing the microorganism (7
days old in PDA) were inoculated under aseptic conditions in pre-
viously autoclaved (121 ◦C for 15 min) 250 mL Erlenmeyer flasks
containing 100 mL of commercial potato dextrose (PD) culture
To a solution of commercial (R)-carvone (1.0 g, 6.67 mmol)
in EtOH (10.0 mL), sodium borohydride (8 equiv.) was slowly

90
F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
medium. After 7 days under static conditions, commercial (R)-
carvone (20, 50, 100, 150 or 250 ␮L) was added to the flasks and
shaken for 3 days at 125 rpm and 28 ◦C. Mycelium samples from
each experiment were replicated on Petri dishes containing PDA
culture medium, and the MIC evaluated after 3 and 6 days of grow-
ing. All of the experiments were performed in six replicates, and
the experiments without carvone were used as a control.
separated from the liquid medium by filtration. In all of the exper-
iments, the liquid medium was extracted with EtOAc (3 × 50 mL)
followed by drying with anhydride Na2SO4 and filtration. The sol-
vent was removed under reduced pressure until ca. 5 mL remained.
The residual volume was increased to 10 mL, and an aliquot of the
solution from each replicate was analyzed by GC–MS. The experi-
ments were performed in duplicate.
2.4.2. Analysis and isolation of the biotransformation products
To the extraction of the biotransformation products from the
experiments with 50 ␮L of (R)-carvone, 30.0 g of NaCl was added to
each replicate, and after 1 h the mycelium was separated from the
liquid medium by filtration. The liquid portion was extracted with
EtOAc (3 × 100 mL) followed by drying with anhydride Na2SO4 and
filtration. The solvent was removed under reduced pressure until
ca. 5 mL remained. The residual volume was increased, to 10 mL,
and an aliquot of the solution from each replicate was analyzed by
GC–MS.
2.6. (R)-carvone biotransformation by growing cells of T.
harzianum
A strain of T. harzianum was cultivated in PDA for 7 days, and
a 3 mm disk of the grown microorganism was inoculated under
aseptic conditions in previously autoclaved (121 ◦C for 15 min)
250 mL Erlenmeyer flasks containing 100 mL of commercial potato
dextrose (PD) culture medium. After 7 days under static condi-
tions, 20 ␮L of commercial (R)-carvone was added to the flasks and
shaken for 3, 6 and 9 days at 125 rpm and 28 ◦C. The mycelium
was separated from the liquid medium by filtration, and the liquid
portion was extracted with EtOAc (3 × 50 mL) followed by drying
with anhydride Na2SO4 and filtration. The solvent was distilled
under reduced pressure until ca. 5 mL remained. The residual vol-
ume was increased to 10 mL, and an aliquot of the solution from
each replicate was analyzed by GC–MS. The experiments were per-
formed in triplicate. Two control experiments were also performed:
flasks with the culture broth and the microorganism (control 1) and
flasks with the culture broth and carvone (control 2).
The remaining solution from one of the replicates was concen-
trated at room temperature, and the crude product (43.0 mg) was
purified via column chromatography over silica gel [100% EtOAc for
6 (6% yield) and 10% EtOAc/MeOH to yield 1a (43.5% yield)].
2.4.3. Confirmation of 1a as a biotransformation product
Isolated compound 6 (2.6 mg) was added to a 100 mL Erlen-
meyer flask with 50 mL of commercial potato dextrose (PD) culture
medium (pH 6.5) and shaken (125 rpm) for 3 days at 28 ◦C. The
material was extracted with EtOAc (3 × 25 mL) followed by drying
with anhydride Na2SO4 and filtration. The solvent was removed
under reduced pressure until ca. 5 mL remained, and the resid-
ual volume was increased to 10 mL. An aliquot of the solution was
analyzed by GC–MS and identified as pure 6.
2.7. Carvone biotransformation by growing cells of M.
circinelloides
2.7.1. Analysis and isolation of the biotransformation products
Because M. circinelloides grows very well in a cleaner basic
medium, such as those based on glucose as carbon sources, bio-
transformation experiments were performed in 47 Erlenmeyer
flasks (250 mL) containing 70 mL of a sterilized Czapeck-type liquid
medium. This medium contained 20 g of glucose, 1.5 g of NaNO3,
0.5 g of KH2PO4, 0.25 g of MgSO4, 0.025 g of FeSO4·7H2O, 0.25 g
of MgSO4 and 2.5 g of KCl dissolved in 1.0 L of distilled water and
20 mg yeast extract. The flasks were autoclaved and inoculated with
pieces of the PDA medium containing mycelium. 6 additional flasks
were maintained as sterility controls. All of the flasks were main-
tained at 28 ◦C in the dark for one day. Next, 20 ␮L of (4S)-carvone
were added to each of the 19 flasks containing the M. circinelloides
culture, and the other 19 flasks were inoculated with 20 ␮L of (4R)-
carvone. The three last flasks, which contained the growing fungus,
were maintained to monitor the metabolites produced by the fun-
gus. All of the flasks were maintained at room temperature for 12
days. After this period of time, the mycelium was separated by fil-
tration, and the liquid phase was extracted with EtOAc (3 × 150 mL)
and n-butyl alcohol (3 × 150 mL). The organic phases were dried
with anhydrous Na2SO4 and distilled under reduced pressure.
The n-butyl extracts was analyzed by TLC and subjected
2.4.4. Kinetic monitoring of the biotransformation of
(4R)-carvone
Two 3 mm disks of the microorganism (7 days old in PDA)
were inoculated under aseptic conditions in previously autoclaved
(121 ◦C for 15 min) 100 mL Erlenmeyer flasks containing 50 mL of
commercial potato dextrose (PD) culture medium. After 7 days
under static conditions, commercial (R)-carvone (20 ␮L) was added
to the flasks and shaken at 125 rpm and 28 ◦C for various time
periods (12, 24 and 48 h). Sodium chloride (30.0 g) was added to
each flask, and after 1 h, mycelium was separated from the liquid
medium by filtration. The liquid portion was extracted with EtOAc
(3 × 50 mL) followed by drying with anhydride Na2SO4 and filtra-
tion. The solvent was removed under reduced pressure until ca.
5 mL remained. The residual volume was increased to 10 mL, and an
aliquot of the solution from each replicate was analyzed by GC–MS.
The experiment was performed in duplicate.
2.5. (R)-carvone biotransformation by the resting cells and
culture broth of L. theobromae
Two 3 mm disks of medium containing the microorganism
(7 days old in PDA) were inoculated under aseptic conditions
in previously autoclaved (121 ◦C for 15 min) 100 mL Erlenmeyer
flasks containing 50 mL of commercial potato dextrose (PD) cul-
ture medium. After 7 days under static conditions, the mycelium
was separated from the culture broth by filtration under aseptic
conditions and suspended in 50 mL of a buffer phosphate (pH 7)
solution. Experiments were performed by the addition of commer-
cial (R)-carvone (20 ␮L) to both the suspended mycelium and the
culture broth. The flasks were shaken at 125 rpm and 28 ◦C for 1, 3
and 6 days. To the experiments with the suspended mycelium, NaCl
(30.0 g) was added to each flask, and after 1 h, the mycelium was
to a sequence of separation processes. The first separation
was performed with a medium pressure chromatography sys-
tem (Combiflash®Rf Teledyne Isco, equipped with RediSep®
C18 275 g ODS column) using linear gradient elution (20–100%
methanol/water in 110 min at a flow rate of 20 mL min−1). A total
of 146 fractions (15 mL each) were collected and analyzed by TLC.
Fractions 61–70 contained only substance 1a, which was analyzed
by NMR (Bruker DRX400 operating at 400 MHz for 1H and 100 MHz
for 13C).
Fractions 45–51 eluted from the CombiFlash system were
also grouped and chromatographed on
a silica gel column.
The separation was performed using isocratic elution with 15%

F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
91
Acetone/Hexane. Twenty-three fractions were collected, and frac-
tion 9 contained (1R,2S,4R,8R)-p-menthane-2,8,9-triol (1b).
(1R,2S,4R,8R)-p-menthane-2,8,9-triol (1b): Yellow amorphous
solid. IR ␯
(cm−1): 754; 840; 954; 994; 1037; 1108; 1143;
max
1299; 1376; 1453; 1663; 2868; 2928; 3392. ESI–MS: m/z 211
([M+Na]+, C10H20O3). 13C NMR (CD3OD, 100 MHz) ı: 19.0(C-7),
21.1(C-10), 28.3(C-5), 29.5(C-6), 34.9(C-3), 37.9(C-1), 38.0(C-4),
69.1(C-9), 71.9(C-2), 75.3(C-8).
2.7.2. Kinetic monitoring of the biotransformation of (4R)- and
(4S)-carvone
M. circinelloides was cultivated in 250 mL Erlenmeyer flasks con-
taining 50 mL of Czapeck medium for 18 h at 28 ◦C under stirring
(125 rpm). Next, 20 ␮L of the substrate ((4R)- or (4S)-carvone) was
added to each Erlenmeyer flask (48 flasks for each carvone). At 4 h
intervals, three flasks were removed, and the mycelium was sep-
arated from the liquid medium. Then, the aqueous portion was
extracted with 20 mL of dichloromethane and 20 mL of n-butyl
alcohol. In the end, 192 samples were obtained (i.e., triplicates of
sixteen samples for each carvone extracted with dichloromethane
and n-butyl alcohol).
3. Results and discussion
3.1. Biotransformation of (R)-carvone by L. theobromae
The study on the biotransformation of carvone was initiated
using whole-cells of the phytopathogen fungus L. theobromae.
The minimum inhibitory concentration (MIC) of (R)-carvone (2)
on the fungal growth was evaluated, and concentrations of 0.2
and 0.5 ␮L/mL of this monoterpene exhibited no inhibition of the
microorganism’s growth. At higher concentrations, (R)-carvone
(2) exhibited either fungistatic (1.0 or 1.5 ␮L/mL) or fungicide
(2.5 ␮L/mL) effects.
The biotransformation products from the culture broth of
the MIC experiments were analyzed by GC–MS. In the experi-
ments performed with monoterpene concentrations of 0.2, 0.5 and
1.0 ␮L/mL, three products, BP-1 (10.6 min), BP-2 (14.4 min) and
BP-3 (15.8 min), were observed besides the substrate 2 (11.3 min).
Their ratio varied in each experiment, and the highest substrate
bioconversion was achieved in the experiments performed with 0.2
and 0.5 ␮L/mL of 2. In the experiments with higher monoterpene
concentrations (1.5 and 2.5 ␮L/mL), only 2 (major compound) and
the product with a retention time of 10.6 min (BP-1) were observed.
Thus, concentrations between 0.2 and 0.5 ␮L/mL of 2 were consid-
ered optimal for the biotransformation of this monoterpene by L.
theobromae.
The
biotransformation
products
extracted
with
dichloromethane were analyzed by GC–MS [Shimadzu CG–MS
2010 plus series equipped with a Restek Rtx® – 5MS capillary
column (30 m × 0.25 mm × 0.25 ␮m)] using helium as the carrier
gas. The oven temperature was programmed at 60 ◦C for 5 min
followed by 60–240 ◦C at 6 ◦C/min and subsequently isothermal
maintained for 5 min. Injector port: 220 ◦C, interface: 240 ◦C, split
ratio: 1:30. The data were acquired using an ionization voltage
of 70 eV. The identification of the compounds was based on mass
spectra by comparison with the NIST Library.n-Butyl alcohol
extracts were analyzed by HPLC–MS/MS. The equipment was
composed of an Alliance 2695 HPLC (Waters, Manchester, UK)
coupled to a Quattro-LC ESI triple-quadrupole mass spectrometer
(Waters, Manchester, UK). The chromatographic separation was
performed on a 4.6 mm × 250 mm Phenomenex Synergy C-18
column (Phenomenex®) with a flow rate of 1 mL min−1. The
chromatographic method employed an isocratic condition with
a mobile phase composed of acetonitrile:water (70:30, v/v) with
0.1% of trifluoroacetic acid (JT Baker). The mass spectrometer
was operated in the ESI positive mode. During these analyses, a
selected ion monitoring experiment (SIM), scanning [M+Na]+ in
both quadrupoles (m/z 211 → m/z 211), was used to monitor the
accumulation of the biotransformation products.
Column chromatography of the crude products from the exper-
iment with 0.5 ␮L/mL of 2 yielded isolated compounds BP-2 (6%
yield) and BP-3 (43.5% yield). Compound BP-1 was not isolated due
to its high volatility.
The main products from the bioreduction of (R)-carvone (2)
are reported in the literature as dihydrocarvone (3) [6,13], carveol
(4) [9] and dihydrocarveol/neodihydrocarveol (5) [6,13]. Therefore,
we decided to chemically reduce (R)-carvone (2) and compare the
products with those obtained from the biotransformation exper-
iment. Analysis of the reaction products by GC–MS allowed the
identification of a mixture of carveol (4) and neodihydrocarveol
(5) with respective retention times of 11.1 and 10.6 min.
A comparison of both the retention time and mass spectra
of neodihydrocarveol (5) with those from the biotransformation
products allowed the identification of BP-1 as 5.
Dihydrocarveol (5): Colorless oil. MS (EI, 70 eV) m/z (%): 154 (3);
136 (72); 121 (84); 107 (97); 93 (100); 79 (84); 67 (63); 55 (59); 41
(75).
8(9)-p-menthen-2,10-diol (6): colorless oil. IR ꢀmax (cm−1): 477;
1034; 1240; 1372; 1451; 1553; 1653; 1723; 2844; 2923; 3399. MS
(EI, 70 eV) m/z (%): 152 (6), 134 (34), 119 (38), 105 (29), 95 (100), 81
(59), 67 (68), 55 (79), 41 (82). 1H NMR (500 MHz, CDCl3) ı: 0.98 (d,
J = 6.7 Hz, H-7); 1.25 (s, H-5ꢀ); 1.47 (m, H-6); 1.47 (ddl, H-3ꢀ); 1.56
(m, H-1); 1.82 (ddd, J = 12.3; 5.7; 3.7 Hz, H-5ꢀꢀ); 1.98 (ddd, J = 13.7;
5.7; 3.2 Hz, H-3ꢀꢀ); 2.38 (ddd, J = 12.4; 5.3; 2.7 Hz, H-4); 3.91 (dl,
J = 2.5 Hz, H-2); 4.15 (s, H-10); 4.90 (s, H-9ꢀ); 5.05 (s, H-9ꢀꢀ). 13C NMR
(125 MHz, CDCl3) ı: 18.5 (C-7); 28.4 (C-6); 32.1 (C-5); 33.9 (C-4);
36.2 (C-1); 39.2 (C-3); 65.4 (C-10); 71.1 (C-2); 108.2 (C-9); 153.7
(C-8).
The biotransformation product BP-2 was identified as
a
diastereoisomeric mixture (de 39.6%) of 8(9)-p-menthen-2,10-diol
(6) using one- and two-dimensional NMR spectra as well as com-
parison with the literature data reported for this monoterpene [19].
The 1H–13C heteronuclear multiple bond correlation (HMBC) spec-
trum of 6 allowed the unambiguous assignment of C-5 (ı 32.1) and
C-6 (ı 28.4), which were reversely assigned in a previous study [19].
Compound BP-3 was identified as (1R,2S,4R,8S)-p-menthane-
2,8,9-triol (1a) by comparison of its NMR data with those reported
in the literature [2].
The hypothesis of (1R,2S,4R,8S)-p-menthane-2,8,9-triol (1a)
being an artifact product formed by the addition of water to the
exocyclic C C bond in 10-hydroxyneodihydrocarveol (6) was con-
sidered. Therefore, biotransformation product 6 was submitted to
the same conditions (culture broth, pH, temperature and time) used
in the biotransformation experiments in the absence of microor-
ganisms. In this case, compound 6 was completely recovered from
the medium confirming 1a as a biotransformation product.
(1R,2S,4R,8S)-p-menthane-2,8,9-triol (1a): Yellow amorphous
solid. IR ꢀmax (cm−1): 754; 840; 954; 994; 1037; 1108; 1143; 1299;
1376; 1453; 1663; 2868; 2928; 3392. ESI–MS: m/z 211 ([M+Na]+,
C10H20O3). MS (EI, 70 eV) m/z (%): 173 (1); 157 (28); 139 (78); 121
(41); 109 (3); 95 (21); 81(51); 43 (100). 1H NMR (500 MHz, CDCl3)
ı: 0.95 (d, J = 6.4 Hz, H-7); 0.99 (d, J = 12.3 Hz, H-5ꢀ); 1.01 (s, H-10);
1.32 (ddd, J = 13.5; 13.4; 2.0 Hz, H-3ꢀ); 1.39 (dd, J = 12.5; 3.2 Hz, H-
6ꢀ); 1.44 (m, H-4); 1.64 (dl, J = 12.3 Hz, H-5ꢀꢀ); 1.99 (brq, J = 12,1 Hz,
H-1); 2.07 (dd, J = 13.4; 2.0 Hz, H-3ꢀꢀ); 3.43 (d, J = 11.3 Hz, H-9ꢀ); 3.55
(d, J = 11.3; H-9ꢀꢀ). 13C NMR (125 MHz, CDCl3) ı: 18.6 (C-7); 19.2 (C-
10); 27.8 (C-5); 28.4 (C-6); 33.5 (C-3); 36.3 (C-1); 36.5 (C-4); 68.8
(C-9); 71.2 (C-2); 75.0 (C-8). 13C NMR (CD3OD, 100 MHz) ı: 19.0(C-
7), 20.7(C-10), 27.3(C-5), 29.5(C-6), 35.9(C-3), 37.9(C-1), 38.0(C-4),
69.2(C-9), 71.8(C-2), 75.4(C-8).

92
F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
CH3
CH3
OH
OH
ii
+
OH
OH
OH
HO
8
8
H3C
OH
CH3
1a
1b
CH3
1
CH3
1
CH3
4R
2
OH
O
2
2
iii
i
HO
CH2
H3C
CH2
H3C
CH2
5
6
5a - (1R, 2S, 4R)
CH3
1
CH3
CH3
OH
O
O
1
2
iv
v
4S
H3C
CH2
H3C
CH2
H3C
CH2
2
3a - (1R, 4S)
5b - (1R, 2R, 4S)
5c - (1R, 2S, 4S)
5d - (1S, 2S, 4S)
3b - (1S, 4S)
Fig. 2. Biotransformation pathway of 4R- and 4S-carvone (2): (i) both C C and C O bonds reduction of (4R)-2; (ii) C C dihydroxylation of 5; (iii) allylic hydroxylation of 5;
(iv) C C bond reduction of (4S)-2; (v) C O bonds reduction of 3a and 3b.
To establish the biotransformation pathway of 2 by L. theobro-
mae, a kinetic experiment (12, 24 and 48 h) was performed under
the same conditions described previously.
In the first 12 h of the experiment, only the starting material (2)
and biotransformation product neodihydrocarveol (5) were iden-
tified in a relative proportion of 28.6 and 71.4, respectively. This
result reveals 5 as the first product formed in the biotransforma-
tion of carvone by L. theobromae. A similar result was also reported
in the literature for the chemical conversion of this monoterpene
by other microorganisms [19].
After 24 h of reaction, both neodihydrocarveol (5) and
(1R,2S,4R,8S)-p-menthane-2,8,9-triol (1a) were the only products
(ratio 1:1), and no (R)-carvone (2) was detected. A comparison of
the results at 24 h with those obtained at 12 h revealed that the
percentage of 5 decreased while the percentage of 1a increased sug-
gesting that compound 1a was formed by the dihydroxylation of 5.
It is important to note that microbial dihydroxylation of the exo-
cyclic double bond of a monoterpene has previously been reported
from the biotransformation of limonene [3].
products revealed that the concentration of 5 decreased while the
concentration of both 1a and 6 increased. These results suggested
that 6 is formed by allylic hydroxylation of 5. However, the for-
mation of 6 from the dehydration of 1a is also possible. Therefore,
a tentative biotransformation pathway of (R)-carvone (2) by the
growing cells of L. theobromae is shown in Fig. 2.
The biotransformation of (R)-carvone by the enzymes from the
culture broth of L. theobromae previously grown for 7 days on BD
medium was also investigated. In this case, the products were ana-
lyzed after 1, 3 and 6 days of reaction. In all experiments, only
neodihydrocarveol (5) was detected as a biotransformation product
with total conversion of (R)-carvone (2) in the sixth day of reaction.
Because (1R,2S,4R,8S)-p-menthane-2,8,9-triol (1a) was not
detected as a biotransformation product in this experiment, the
enzymes responsible for the conversion of 2 into 1a were expected
to be present in the mycelium of the fungus. To verify this hypoth-
esis, a new experiment was performed using the resting cells of
L. theobromae, which were previously grown for 7 days on BD
medium, suspended in a phosphate buffer at pH 7. The biotransfor-
mation products were analyzed after 6 days of reaction, and only
neodihydrocarveol (5) was detected. Therefore, the formation of 1a
occurs only when growing cells of the fungus are used.
The results obtained at 48 h of reaction indicated the pres-
ence of biotransformation products 5, 6 and 1a (respective ratio
6.5:1.0:2.5). In this experiment, the relative composition of the

F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
93
Fig. 3. (A) Estimated consumption of the (4S)-carvone (2) and accumulation of its reduction products (5b, 5c, and 5d) over 60 h of fermentation; (B) kinetic profile of
production of menthanetriols 1a and 1b by M. circinelloides. The error bars correspond to standard deviation of biological replicates (triplicates).
3.2. Biotransformation of (R)-carvone by T. harzianum
and (4S)-neo-dihydrocarveol (5c, RI: 1196), which formed from 3a
(Fig. 2).
The growing cells of T. harzianum, an antagonist fungus from
soil used in the control of some phytopathogens [20], were also
investigated as source of enzymes for the biotransformation of (R)-
carvone (2).
In contrast to the results with the L. theobromae whole-cells,
only biotransformation product 5 was identified in the experiments
with T. harzianum. Since the third day of the reaction, 5 was the
major product (84.2%), and its concentration continued to increase
until the 9th day of reaction where it reached 98.6%.
These results indicated that the biotransformation of (R)- and
(S)-carvone by M. circinelloids follows different pathways. For (R)-
carvone, the first biotransformation step is the stereoselective
reduction of the double bond and carbonyl groups by enone reduc-
tase and carbonyl reductase, respectively [1,15]. These reactions
convert (R)-carvone (2) into (1R,2S,4R)-dihydrocarveol (5a). The
last step on the (R)-carvone biotransformation pathway should be
the dihydroxylation of the remaining double bond resulting in tri-
hydroxylated menthanetriols (1a and 1b). Because dihydrocarveol
(5a) is the biosynthetic precursor of menthanetriols (Fig. 2) in the
proposed biotransformation pathway, the stereogenic centers at C-
1 and C-2 in 5 are not involved in the biotransformation process.
Therefore, both menthanetriols (1a and 1b) and dihydrocarveol
(5a) must have the same absolute configuration at these stereo-
centers.
3.3. Biotransformation of (R)- and (S)-carvone by M.
circinelloides
The use of cells from M. circinelloides for the biotransformation
of terpenes has been reported in the literature [20]. A strain of this
fungus was isolated from the wood of Pinus taeda, a plant that is
a good producer of monoterpenoids including carvone. Therefore,
its potential for the biotransformation of (R)- and (S)-carvone was
also investigated. During the analyses of n-butyl alcohol extract
by TLC, some biotransformation products from (R)-carvone (2)
were observed. Then, after a sequence of chromatographic pro-
cedures, two substances were isolated and analyzed by mass and
NMR spectroscopy. By comparing their spectrometric data with
those published in the literature [1,2], it was possible to identify
compound 1a as (1R,2S,4R,8S)-p-menthane-2,8,9-triol and 1b as
(1R,2S,4R,8R)-p-menthane-2,8,9-triol (Fig. 2).
The ethyl acetate extracts from the (R)-carvone biotransfor-
mation experiment were analyzed by GC–MS to identify the
biotransformation products by comparison with the NIST library.
After 4 h of fermentation, the extract from the (R)-carvone bio-
transformation was primarily composed of one product that was
identified as neodihydrocarveol (5a, 94%), which is presumed to be
the precursor of menthanetriols 1a and 1b (Fig. 2).
For (S)-carvone (2), the biotransformation pathway is different,
and the first biotransformation products include the (1R,4S)-
dihydrocarvone (3a) and (1S,4S)-dihydrocarvone (3b) epimers in a
9:1 ratio after 4 h of fermentation. These compounds result from the
regioselective reduction of the endocyclic double bond by enone
reductase. During the next biotransformation step, 3a is converted
into a nearly 1:1 mixture of the two (4S)-dihydrocarveol stereoiso-
mers (5b and 5c) by carbonyl reductase.
3.3.1. Kinetic monitoring of (R)- and (S)-carvone consumption
and menthanetriol production
After 4 h of biotransformation reaction, the concentration of
both R- and S-carvone drops to a nearly undetectable amount. The
products formed were identified as shown above. These experi-
ments were extended to cover 60 h of fermentation to investigate
the interconversion of the intermediates to products. The studies
were conducted in triplicate, and samples were collected every
four days. The dichloromethane extracts were analyzed by GC–MS,
while the n-butyl extracts were analyzed by LC–MS/MS.
A similar investigation was also performed in parallel using (S)-
carvone. Analyses of the n-butanol extract using LC–MS/MS showed
that no menthanetriol was produced from this carvone enantiomer
by M. circinelloides. The GC–MS analysis of the ethyl acetate extracts
indicated that (S)-carvone is almost completely consumed after 4 h
of fermentation yielding (1R,4S)-dihydrocarvone as the main prod-
uct (3a, 75%, Retention Index: 1209). The other dihydrocarvone
diastereoisomer (1S,4S; 3b) is also produced along with minor com-
pounds that were identified as (4S)-dihydrocarveol (5b, RI: 1217)
After 4–24 h of fermentation, the four peaks in the GC–MS chro-
matogram correspond to products from (S)-carvone (3a and its
reduced products 5b and 5c; and 3b). During this period, 3a is trans-
formed to 5b and 5c, which is observed by a drastic decrease in its
peak area and enhancement of the 5b and 5c peaks. After 24 h, a
shoulder appears in the 3b peak (R.T. 15.00 min) and is completed
formed as a chromatographic peak (R.T. 14.92 min) after 60 h. This
peak corresponds to 5d. These transformations are illustrated in
Fig. 3A. Overall, a high percentage of (1R,4S)-dihydrocarvone (3a)

94
F.M. Nunes et al. / Applied Catalysis A: General 468 (2013) 88–94
is obtained during the first few hours, and the carveols tend to
accumulate with no diastereoselectivity after 60 h.
Acknowledgments
The biotransformation of (R)-carvone over 60 h primarily
produces dihydrocarveol (5a). The conversion of (R)-carvone into
this compound occurs with 94% conversion in the first 4 h. Dur-
ing the next few hours, the concentration of 5a slightly decreased,
and after 8 h of culture, the production of both stereoisomers of
menthanetriols (8R and 8S) begins and continues until 48 h when
it starts to decrease by an unknown process (Fig. 3B).
The authors wish to acknowledge the Fundac¸ ão de Amparo
à Pesquisa do Estado de São Paulo (FAPESP), Fundac¸ ão Cearense
de Apoio ao Desenvolvimento Científico e Tecnológico (FUN-
CAP), Conselho Nacional de Desenvolvimento Científico e Tec-
nológico (CNPq), Programa Sisbiota-Brasil, and Coordenac¸ ão
de Aperfeic¸ oamento de Ensino Superior (CAPES) for financial
support.
The transformation of (R)-carvone (2) into (1R, 2S, 4R)-
dihydrocarveol (5a) in only 4 h and 94% yield may be of industrial
interest since dihydrocarveol is used as flavoring food additive and
it is worth five times more than carvone (2) [21]. Additionally, the
production of 5 (mixture of isomers) from carvone has been carried
out by chemical reagents, and environmentally friendly method of
its production would be desirable.
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