48
H. Kihara et al. / Phytochemistry 107 (2014) 42–49
respectively. The mass detector was operated in the electron
impact mode with an ionization energy of 70 eV.
crude enzyme soln. The reaction was initiated by mixing the crude
enzyme soln. (200 L) suspended in
L) with 10 mg mLÀ1 3 (10
l
l
To identify each compound, retention indices and MS profiles of
corresponding authentic specimens were used. To construct cali-
bration curves for quantification, a cotton swab containing given
amounts of 1 (Wako Pure Chemicals, Osaka, Japan), 3 (Sigma–
Aldrich, St. Louis, MO), 4 (Tokyo Chemical Industry Co., Tokyo,
Japan), 2 (Tokyo Chemical Industry), 5 (Alfa Aesar, Ward Hill,
MA), and caryophyllene (Sigma–Aldrich) was placed approxi-
mately 2–3 cm above ground with almost the same soil composi-
tion as the collection site but without any plants; then, the
ground was covered with the hood for SPME collection. An aqueous
soln. of each compound was prepared by suspending it in a small
amount of Tween 20. In the absence of addition of the volatile com-
pounds, the emission of the volatiles was not detected.
0.2% Tween 20 in 50 mM Na phosphate, pH 7.0 (total volume of
1 mL) in a shaking water bath at 24 °C for 30 min. After the reac-
tion, the C8 compounds were extracted with MTBE and analyzed
using GC.
Resolution of each enantiomer was carried out using a GC
equipped with a 0.25 mm  30 m
a-DEX 120 column (Supelco).
The column temp. was maintained at 100 °C. Injector and detector
(FID) temperature was set at 200 °C. N2 gas was used as carrier gas
at 20.0 cm sÀ1. Under these conditions, (S)-5 and (R)-5 (Acros
Organics, Geel, Belgium) eluted at 11.0 and 11.2 min, respectively.
4.5. Acetylation activity
To follow the time course after mechanical wounding, the lawn
of thalli was gently covered with a round-shaped polystyrene cup
(basal area, 58.1 cm2; height, 5 cm) immediately after mechanical
wounding to 10% of the surface, and the headspace inside of the
cup was continuously passed through a charcoal cartridge (ORBO
32 Small Activated Coconut Charcoal (20/40), 100/50 mg, Sigma–
Aldrich) at 1000 mL minÀ1 with an air-pump (N86KT.18, KNF Japan
Co., Tokyo, Japan). After collecting volatiles for 40 min, the
adsorbed compounds were eluted three times with CH2Cl2
To examine the acetylation activity responsible for forming 3
from 5, the des6KO mutant strain was used. The des6KO thalli grown
on a half-strength Gamborg-B5 plate were exposed to the vapor of
5 by hanging a sheet (7 Â 7 mm) of filter paper containing
10 mg mLÀ1 5 soln.(20
l
L) in 0.2% Tween 20 for 18 h under contin-
uous light at 21 °C. As a control, 0.2% Tween 20 (20
l
L) was used.
After exposure, the thalli were collected from the plate, and the
volatiles in the tissues were immediately extracted with MTBE as
described in Section 4.3.
(250 lL containing 1 l
g mLÀ1 cyclohexanol). A portion of the
extract was subjected to GC–MS analysis essentially as shown
above. Injection was performed using a split-mode with a ratio
of 10. The column temperature was programmed as follows:
40 °C for 2 min, increasing by 5 °C minÀ1 to 150 °C, by 10 °C minÀ1
to 200 °C, then held at 200 °C for 2 min. The carrier gas (He) was
delivered at 86.1 kPa.
4.6. Oxidoreductase assay
The thalli were collected and incubated on wet paper towel for
3 h at 24 °C to recover from the response caused by wounding dur-
ing collection. The thalli (ca. 0.1 g fr wt) were mixed with an equal
volume of distilled H2O with or without 5 mM NADH or NADPH,
and then were thoroughly homogenized in a mortar. The homoge-
nate was transferred into a glass vial (22 mL, Perkin Elmer, Wal-
tham, MA, USA), sealed tightly with a butyl rubber stopper and a
crimp-top seal (National Scientific, Rockwood, TN, USA), and the
volatiles in the headspace were collected using a SPME fiber at
25 °C for 30 min. To analyze volatiles from partially wounded
thalli, about 50% of the surface area of thalli were mechanically
wounded with forceps, and then placed in the vial for SPME collec-
tion. Intact thalli were used as the control. The volatiles were ana-
lyzed as described in Section 4.2. Aq. solutions of standard
compounds were prepared from 10 mg mLÀ1 suspensions in 0.2%
Tween 20 and used to construct calibration curves.
4.3. Solvent extraction
The thalli collected from the campus and grown in a growth
chamber for 3 months were used for solvent extraction. Three
hours before analysis, the thalli were collected and placed on a
wet towel to avoid wound-responses brought about during collec-
tion. The thalli were snap-frozen with liquid N2, and powdered
with a mortar. Volatiles were extracted from a portion (0.2 g fr wt)
with 1 mL of methyl tert-butyl ether (MTBE) containing nonanyl
acetate (1 l
g mLÀ1) (Tokyo Chemical Industry). Another portion
(0.2 g fr wt) was thawed in a water bath, and then incubated for
an additional 5 min at 24 °C to facilitate the enzyme reaction.
The enzyme reaction was terminated by adding MTBE (1 mL) con-
taining nonanyl acetate. The MTBE mixture was dispersed with the
aid of an ultrasonic washer for 10 s, then mixed vigorously. The
mixture was centrifuged at 210Âg for 10 min at 25 °C (8100 Centri-
fuge, Kubota, Tokyo, Japan). A portion of the extract was subjected
to GC–MS analysis essentially as described above. Injection was
performed using a split-mode with a ratio of 2. The column tem-
perature was programmed as follows: 40 °C for 2 min, increasing
by 5 °C minÀ1 to 200 °C for 2 min. The carrier gas (He) was deliv-
ered at 26.7 cm sÀ1. For quantification of 1, 2, 3, 4, and 5, each stan-
dard was suspended in 0.2% Tween 20 at 10 mg mLÀ1, and the
suspension was diluted to prepare a series of aq. solutions,
extracted with MTBE containing nonanyl acetate, and then sub-
jected to GC–MS analysis to construct calibration curves.
To estimate the oxidoreductase activity toward 5, crude enzyme
soln. (100
l
L) prepared as described in Section 4.4. was mixed with
10 mg mLÀ1 5 (10
lL) suspended in 0.2% Tween 20, and incubated
at 27 °C for 5 min in 50 mM sodium phosphate (pH 7.0) with or
without 5 mM NADH or NADPH. After the reaction, the C8 com-
pounds were extracted with 1 mL of MTBE containing nonanyl ace-
tate (1 l
g mLÀ1), and quantitatively analyzed using GC–MS
essentially as described in Section 4.4 but with the modified col-
umn condition [40 °C (5 min) to 200 °C (2 min) at 8 °C minÀ1 with
He as a carrier gas at 26.7 cm sÀ1].
4.7. Fatty acid analysis
Approximately 2 g (FW) of M. polymorpha thalli were ground at
room temperature and extracted with CHCl3: MeOH (9 mL, 1:2 v/v)
for 20 min. After adding 5 mL each of CHCl3 and H2O, the organic
phase was recovered by centrifugation at 1860Âg, at 4 °C for
20 min. After addition of EtOH (À15 mL), the extract was freeze-
dried, and fatty acids were trans-methylated with 10% (v/v)
H2SO4 in MeOH (2.5 mL) at 80 °C for 2 h. Fatty acid methyl esters
were extracted with hexane (2.5 mL), and a portion was analyzed
with GC–MS (Shimadzu GCMS-QP2010 Ultra) equipped with a
quadrupole mass detector (EI, 70 eV) and a DB-23 capillary column
4.4. Esterase assay
The thalli (wild type or des6KO, 5 g fr wt) were homogenized
with 50 mM Na phosphate (10 mL, pH 7.0 containing 2.5% Polyklar
VT (w/v)) (Wako Pure Chemicals) in a mortar with the aid of sea
sand on ice. The homogenized soln. was centrifuged at 10,000Âg
for 30 min at 4 °C, and the resultant supernatant was used as the
Kihara, Hirotomo
