1366
S. ODA and K. ISSHIKI
and L-L IBRs. Concerning the suspension and two-
liquid-phase systems, 1 ml of a 3-d broth was inoculated
to 50 ml of the liquid medium prepared in a 250-ml
flask. After 1 d of precultivation at 25 ꢁC, 5 ml of a 20%
solution of benzil in DMSO and 250 ml of Tween-80
(suspension system) or 10 ml of a 1% solution of benzil
in hexyl ether (two-liquid-phase system) were added to
the 1-d broth, and cultivation was done at 25 ꢁC with
shaking (220 rpm) for 4 d. As for the S-L IBR, 200 ml of
a 3-d broth was inoculated on the surface of a modified
Sabouraud agar plate (volume, 10 ml; surface area,
7.1 cm2) prepared in a glass vial (volume, 50 ml;
diameter, 3 cm). Following stationary precultivation for
1 d, 2 ml of a 2% solution of benzil in hexyl ether was
added onto a fungal mat formed during precultivation,
and cultivation was done at 25 ꢁC without shaking for
4 d. As for the L-L IBR, 200 ml of a 3-d broth was
inoculated to a liquid medium containing 200 mg of MS
(MFL-80GCA; CaCO3-coated type; mean diameter,
20 mm; density, 0.2; Matsumoto Yushi-Seiyaku, Osaka),
and precultivation was done at 25 ꢁC without shaking for
1 d after vigorous mixing the inoculated mixture. After
stationary precultivation, 2 ml of a 2% solution of benzil
in hexyl ether was applied onto a MS-fungus mat formed
during the precultivation, and cultivation was done at
25 ꢁC without shaking for 4 d. The concentrations of
benzil, (S)-benzoin, and hydrobenzoin in ethyl acetate
extract from the broth (suspension system) or hexyl
ether layer (two-liquid-phase, S-L, and L-L IBR sys-
tems), and ee of (S)-benzoin were determined by above-
mentioned methods.
20
18
16
14
12
10
8
6
4
2
Benzil
(S)-Benzoin
Fig. 2. Comparison of the Benzil-Reduction Activities among
Suspension, Organic-Aqueous Two-Liquid-Phase, S-L, and L-L
IBR Systems.
As for the suspension system, 5 ml of a 20% solution of benzil in
DMSO and 250 ml of Tween-80 were added to 50 ml of 1-d broth. As
for the two-liquid-phase system, 10 ml of a 2% solution of benzil in
hexyl ether was added to 50 ml of 1-d broth. Both systems were
applied to the reduction of benzil at 25 ꢁC with shaking (220 rpm).
As for the S-L and L-L IBRs, 2 ml of a 2% solution of benzil in
hexyl ether was added onto a fungal and a fungus-MS mat (1 d of
precultivation; surface area, 7.1 cm2), respectively. The reduction
was performed at 25 ꢁC without shaking.
Acknowledgment
As shown in Fig. 2, the reduction of benzil did not
proceed significantly in the suspension system due to the
toxicity of benzil. The two-liquid-phase and S-L IBR
systems exhibited moderate reducing activities to afford
9.6 (86.1% ee) and 10.2 g/l of the organic phase (96.8%
ee) of (S)-benzoin, respectively. It was assumed that
hexyl ether layer served as a reservoir for the toxic
benzil and (S)-benzoin produced. Furthermore, the
L-L IBR exhibited more excellent reducing activity
than two-liquid-phase and S-L IBR systems, to afford
14.4 g/l of the organic phase of (S)-benzoin (99.0% ee).
Thus the L-L IBR exhibited excellent performance in
the asymmetric reduction of benzil to (S)-benzoin, in
addition to the other bioconversion, the hydrolysis of
2-ethylhexyl acetate.13) In addition to the high accumu-
lation of hydrophobic products, easier fungal morphol-
ogy control, a higher supply of nutrients, water, and
oxygen, and a lower demand for energy are superior
merits, as compared with traditional fungal cultivation
systems, submerged, solid-state, and liquid-surface
cultivation systems.
We express our sincere thanks to Director Nobuo
Ichimaru, Matsumoto Yushi-Seiyaku, Co., Ltd., for the
kind gift of a microsphere. We are also grateful to Miss
Aki Kobayashi for helpful technical assistance.
References
1) Chartrain, M., Armstrong, J., Katz, L., Keller, J., Mathre,
D., and Greasham, R., Asymmetric bioreduction of a ꢀ-
ketoester to (R)-ꢀ-hydroxyester by the fungus Mortier-
ella alpina MF 5534. J. Ferment. Bioeng., 80, 176–179
(1995).
2) Salvi, N. A., and Chattopadhyay, S., Rhizopus arrhizus
mediated asymmetric reduction of alkyl 3-oxobuta-
noates. Tetrahedron: Asymmetry, 15, 3397–3400 (2004).
3) Demir, A. S., Hamamci, H., Ayhan, P., Duygu, A. N.,
Igdir, A. C., and Capanoglu, D., Fungi mediated
conversion of benzil to benzoin and hydrobenzoin.
Tetrahedron: Asymmetry, 15, 2579–2582 (2004).
4) Konishi, J., Ohta, H., and Tsuchihashi, G., Asymmetric
reduction of benzil to benzoin catalyzed by the enzyme
system of a microorganism. Chem. Lett., 1985, 1111–
1112 (1985).
However, it was observed that excess (S)-benzoin
produced accumulated as many needle-like crystals on a
fungus-MS mat. This practically unfavorable phenom-
enon must be overcome by the construction of an in situ
product removal system, such as the adsorption or
crystallization of (S)-benzoin.8)
5) Ohta, H., Konishi, J., Kato, Y., and Tsuchihashi, G.,
Microbial reduction of 1,2-diketones to optically active
ꢁ-hydroxyketones. Agric. Biol. Chem., 51, 2421–2427
(1987).
6) Saito, T., Maruyama, R., Ono, S., Yasukawa, N.,
Kodaira, K., Nishizawa, M., Ito, S., and Inoue, M.,