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