R. Gandolfi et al. / Tetrahedron: Asymmetry 12 (2001) 501–504
503
Temperature did not exert a strong influence on the
stereoselectivity of Aspergillus oryzae, while almost
complete enantioselectivity was obtained with Rhizopus
oryzae at lower temperatures. It is likely that more than
one enzyme of the mycelium can catalyze the esterifica-
tion of 2-phenylpropanoic acid, but only the highly
enantioselective ones are really active at lower
temperatures.
It can be concluded that dry mycelium of Aspergillus
oryzae MIM and Rhizopus oryzae CBS 112-07 can be
used as enantioselective biocatalysts for the production
of enantiomerically enriched ethyl (R)- or (S)-2-phenyl-
propanoate in organic solvents. Appropriate choice of
solvent and temperature allowed for the formation of
the (R)-ester as the only enantiomer using Rhizopus
oryzae.
hydrogen flame ionization detector; the column temper-
ature was kept at 180°C. The column (3×2000 mm) was
packed with Carbowax 20 M (10% 100/120 mesh,
Supelcoport). The absolute configuration of the
obtained esters was determined by comparison with the
specific rotation of authentic samples of the pure enan-
tiomers obtained by esterification of enantiomerically
pure (S)-2-phenylpropanoic acid with different alcohols
using conventional esterification procedures.5 The enan-
tiomeric composition was routinely determined by gas
chromatographic analysis of the esters using a chiral
capillary column (diameter 0.25 mm, length 25 m,
thickness 0.25 mm, DMePeBeta-CDX-PS086, MEGA,
Legnano, Italia). For ethyl, propyl and butyl 2-phenyl-
propanoates the column temperature was kept at 90°C
for 15 min and then 1°C/min; retention times: (R)-ethyl
ester 26.8; (S)-ethyl ester 27.5; (R)-propyl ester 35.5;
(S)-propyl ester 36.2; (R)-butyl ester 35.8; (S)-butyl
ester 36.5 min. For 2-phenylpropanoic pentyl and hexyl
esters the column temperature was kept at 90°C for 10
min and then 1°C/min; retention times: (R)-pentyl ester
30.7; (S)-pentyl ester 31.4; (R)-hexyl ester 34.9; (S)-
hexyl ester 35.6 min. For isoamyl 2-phenylpropanoate
the column temperature was kept at 80°C for 15 min
and then 1°C/min; retention times: (R)-isoamyl ester
44.1; (S)-isoamyl ester 27.5 min. The stereochemical
outcome of the transformations was expressed as enan-
tiomeric excess (e.e.) of the major enantiomer or as the
enantiomeric ratio (E).6,7
3. Experimental
3.1. Microorganisms, growth and biotransformation con-
ditions
Aspergillus oryzae MIM (Microbiologia Industriale
Milano) and Rhizopus oryzae CBS (Centraal Bureau
voor Schimmelcultures, Baarn, Holland) 112.07 were
used throughout this study and routinely maintained on
malt extract (8 g L−1, agar 15 g L−1, pH 5.5). The
microorganisms were cultured in 500 mL Erlenmeyer
flasks containing 100 mL of medium and incubated for
48 h at 28°C on a reciprocal shaker (100 spm). The
liquid media contained a basic medium (BM: Difco
yeast extract 1 g L−1, (NH4)2SO4, 5 g L−1, K2HPO4, 1 g
L−1, MgSO4·7H2O, 0.2 g L−1, pH 5.8) supplemented
with Tween 80 (0.5%). Suspensions of spores (1.6×104)
were used as inoculum. The microorganisms were also
cultured in a 10 L stirred tank reactor containing 2 L of
medium at 28°C, 200 rpm and aeration 1 vvm. Cells
grown for 48 h in submerged cultures were harvested by
filtration at 4°C, washed with phosphate buffer (pH 7.0,
0.1 M) and lyophilized. Ester synthesis was carried out
in 10 mL screw capped test tubes by suspending
lyophilized mycelium in organic solvent (5 mL) and then
adding the alcohol and the acid. The reaction mixtures
were magnetically stirred at different temperatures.
Acknowledgements
This work was supported by the C.N.R. Target Project
on Biotechnology (n 97.01019. PF 115.08601).
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Gandolfi, Raffaella
