A. Rajagopalan et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 118–122
119
O
+
was diluted with water and tested for glucose concentration using
the CONTOUR TS.
crude enzyme preparation
Trametes hirsuta
H
O
2.2.6. Cell dry weight determination
buffer, pH 6.0
The CDW was determined using an automatic thermobalance
(Sartorius MA 30 moisture analyzer). The filtered cells of 5 mL cul-
25 °C, 24 h
H
◦
MeO
ture were heated until constant weight at 130 C.
MeO
1
2
2bar O2
p-anisaldehyde
2.2.7. Optimized cultivation conditions
t-anethole
The preculture (100 mL sorbix medium in 250 mL flask), inocu-
2
Scheme 1. Biocatalytic alkene cleavage of t-anethole to p-anisaldehyde by T. hirsuta.
lated by a piece of agar with fungus (1 cm ) was grown for 5 days at
◦
2
5 C and 150 rpm. The sample (16.5 mL) was used to seed the main
culture (330 mL in 1000 mL non-baffled flask) which was incubated
at 25 C and 150 rpm. After 24, 48 and 72 h of growth, t-anethole
2.2. Methods
◦
(
100 L of 50 mM stock in ethylacetate) was added to the culture
2
.2.1. Growth medium
The growth medium for T. hirsuta FCC 047, the “sorbix”
medium. After 96 h, the dosage was increased (300 L of 50 mM
stock in ethylacetate). The culture was finally harvested after 100 h
of cultivation.
medium, contained glucose (15 g/L), l-asparagine monohydrate
(
(
3 g/L), KH PO (1.5 g/L), MgSO ·7H O (0.5 g/L), yeast extract
2
4
4
2
6 g/L), 3,4-dimethoxybenzyl alcohol (0.1 mL) and 1 mL of trace
2
.2.8. Biotransformations
For the determination of the conversion, a previous procedure
element solution SL4 [EDTA (0.5 g/L), FeSO ·7H O (0.2 g/L),
4
2
ZnSO ·7H O (0.1 g/L), MnCl ·4H O (0.03 g/L), H BO (0.3 g/L),
4
2
2
2
3
3
[
25–27] was adapted. Lyophilized cells (30 mg) were resuspended
CoCl ·6H O (0.2 g/L), CuCl ·2H O (0.01 g/L), NiCl ·6H O (0.02 g/L),
2
2
2
2
2
2
in Bis–Tris buffer (1 mL, 50 mM, pH 6.0). The cells were ultraso-
nicated (1 s pulse on, 4 s pulse off for a total time of 100 s, 50%
Na MoO ·2H O (0.03 g/L)].
2
4
2
◦
amplitude) and centrifuged (4 C, 8000 rpm, 20,000 × g, 20 min).
2.2.2. Strain maintenance
The supernantant was used for the assay performed in 48 well
RP plates. Each sample was tested in duplicates. The reaction mix-
ture contained the cell free extract (900 L) and t-anethole (5 L,
T. hirsuta FCC 047 was maintained on agar plates of sorbix
◦
medium (with 18 g/L of agar). The plates were stored at 4 C. Addi-
tionally cells were stored as cryostocks of sorbix and a cryo solution
4
.94 mg, 33 mol). In case of other tested substrates, an additional
[
sodium glutamate (50 g/L), saccharose (50 g/L), dextrane (50 g/L),
co-solvent (DMSO, 100 L, 10%, v/v final concentration) was also
added to improve solubility [28]. The reaction was run under oxy-
gen pressure (2 bar) and shaken (170 rpm, GFL 3015 orbital shaker)
pH 6.4] in a ratio of 1:1.
2.2.3. Growth study
◦
at 21 C. After 24 h, the samples were extracted with ethyl acetate
For the preculture sorbix medium (100 mL) in non-baffled flasks
(2× 500 L) and analysed by GC.
(
300 mL) was used while for the main cultures 1 L non baffled flasks
The concentrations of the t-anethole and p-anisaldehyde were
were employed (500 mL medium). The conditions for growth were
2
determined using a calibration curve. n-Decane was used as an
internal standard in the solvent (ethyl acetate) which was used for
the extraction of the samples.
◦
2
5 C and 150 rpm on a rotary shaker. A piece of agar (1 cm ) with
the fungus was used to seed the preculture and grown for 5 days.
A fraction of the preculture (25 mL) was employed for seeding the
main cultures. In case of the culture grown in the presence of t-
anethole, t-anethole (200 L, dissolved in DMSO, 1 M stock) was
added to set the concentration of t-anethole to 0.4 mM at 24 h. The
same amount was also added after 48, 73 and 97 h of cultivation.
The culture in the presence of t-anethole vapour was grown just
beside the flask containing t-anethole. The flask in the absence of
t-anethole was grown separately. Culture samples (50 mL) were
withdrawn from all the three flasks after 24, 48, 73, 97 and 122 h.
A fraction (5 mL) was used for cell dry weight determination and
the remaining (45 mL) was prepared for the biotransformation (see
Section 2.2.4).
3
. Results
3
.1. A fungicide as inducer
Literature suggested that t-anethole 1, which was actually the
best accepted substrate for alkene cleavage by T. hirsuta, is a very
effective fungicide component present in essential oils produced
by certain plants [29–31]. Therefore, we speculated that the fun-
gus has developed the alkene cleavage activity in a response to
this natural fungicide or related ones. To test the fungicide activ-
ity, t-anethole 1 was added to the liquid growth medium of the T.
hirsuta (final concentration of 13.2 mM in 100 mL medium) which
was inoculated with a sample of the fungus, which previously
showed low alkene cleavage activity (17% conversion of t-anethole
to p-anisaldehyde under standard reaction conditions). In parallel a
control experiment was run in the absence of t-anethole. After 24 h,
it was observed that the control flask was growing well, whereas
the fungus in the presence of 1 did not grow. The minimal inhibitory
concentration of t-anethole for the white-rot fungi Trametes ver-
sicolor was reported to be 1.25 mM [31]. Keeping this number in
mind, the subsequent experiment was performed by a daily addi-
tion of a lower concentration of t-anethole (0.4 mM) to the growth
medium right from the beginning. The daily addition was required
due to the high volatility of t-anethole.
2.2.4. Lyophilization of cells
The culture samples (45 mL each) were centrifuged (8000 rpm,
◦
1
9,945 × g, 4 C, 20 min). A fraction of the supernatant (1 mL)
was taken for glucose determination. The remaining supernatant
was discarded and the pellet was resuspended in Bis–Tris buffer
(
∼50 mL, pH 6, 50 mM) for washing. After centrifugation, the pellet
obtained was lyophilized and used for the biotransformation.
2.2.5. Glucose determination
CONTOUR TS (Bayer), a blood sugar measuring device, was used
for determination of the concentration of glucose in the fungal
growth medium. To adapt the protocol for glucose in the growth
medium, a calibration curve (y = 0.148x, correlation coefficient
R = 0.98) showed linearity between 0 and 15 mg/mL. The super-
natant after the first centrifugation step of the collected samples
It was observed that after seven days of cultivation, the culture in
the presence of 1 displayed lower final cell yield after lyophilization
compared to the control flask. However, in the biotransformation
2