R.-C. Zheng et al. / Catalysis Communications 18 (2012) 68–71
69
2.5. Gas chromatography analysis
Enantiomeric compositions of residual amide and the corresponding
acid were determined by gas chromatography on a γ-cyclodextrin
based chiral column BGB-175 [27]. Retention times for (S)-1, (R)-1,
(
R)-2, and (S)-2 were 9.10 min, 9.30 min, 7.82 min and 8.65 min,
respectively.
The enantiomeric ratio (E) and concentration of the four enantio-
S P
mers in the reaction mixture were calculated based on ee and ee as
the method developed by Rakels and us [28,29].
Scheme 1. Amidase-mediated stereospecific hydrolysis of (R,S)-1 to (S)-1, a key segment
of cilastatin.
3
. Results and discussion
from Huadong Medicine Group (Hangzhou, China). The other chemi-
cals used in this work were of analytic grade from local suppliers.
3.1. Selection of cosolvents added to the reaction mixture
It has been reported in many cases that addition of water-miscible
organic solvent can enhance the enantioselectivity and activity of bio-
catalysts [30], especially in hydrolase (lipase, esterase and protease)
catalyzed reactions [18,19]. In this work, effects of 8 cosolvents on
both catalytic activity and enantioselectivity of the amidase were
first studied at a concentration of 10% (v/v). As shown in Fig. 1, addi-
tion of acetonitrile, ethanol, methanol and acetone significantly accel-
erated initial rate of the hydrolytic reaction. In particular, amidase
activity was enhanced to 52.9 and 46.2 U/g cdw in the presence of
acetonitrile and ethanol, which was 3.7 and 3.2 times higher than
that in the neat aqueous buffer (14.3 U/g cdw). With regard to enan-
tioselectivity, cells of D. tsuruhatensis only exhibited moderate enan-
tiomeric ratio (E=27) to (R,S)-1 while a notable increase (E>80)
was observed upon the addition of acetonitrile, ethanol, and DMSO.
Because catalytic activity was much lower in the presence of DMSO,
acetonitrile and ethanol were selected as optimal additives for further
investigation.
2
.2. Microorganisms and cultivation conditions
D. tsuruhatensis ZJB-05174 was isolated through a colorimetric high-
throughput screening system [25]. The cell culture of D. tsuruhatensis
ZJB-05174 was performed in 250 ml flasks with 40 ml of sterile medium
containing (g/l): glucose 8.4, acetamide 3.56, yeast extraction 6.3, pep-
tone 0.7, NaCl 1.0, KH PO 1.0, and K HPO 1.0 (pH 7.5). The cells were
2 4 2 4
harvested after 20 h of incubation (150 rpm) at 30 °C by centrifugation
at 9000×g for 10 min and stored at 4 °C for further use.
2
.3. Biotransformation
Amidase-catalyzed hydrolysis of 1 was carried out at 30 °C in
shaking flasks on an orbital shaker at 120 rpm. The reaction mixture
consist of 4.5 ml Tris–HCl buffer (50 mM, pH 8.2), 30 mM (R,S)-1,
0
.5 ml different organic solvent and 0.3 g wet resting cells (approxi-
mately 27 mg cell dry weight (cdw)). Samples (0.5 ml each) were
withdrawn after 10 min and the reaction was quenched by addition
of 30 μl HCl (5 M). After centrifugation, 200 μl supernatant was
extracted with 800 μl ethyl acetate. The ethyl acetate layer was
2
Solubility of 1 in aqueous solution was low (0.54 g/100 ml H O,
30 °C), but it was almost doubled with the addition of cosolvents,
e.g. 12.7 g/l in the presence of 10% EtOH. The increase of substrate
concentration and reduced mass-transfer resistance from membrane
and wall of whole cells contributed to enhancement of amidase activ-
ity. The mechanism responsible for significant improvement of enan-
tioselectivity after addition of cosolvents was more complicated and
might lie in two aspects. Firstly, specific interactions probably exist
between the amidase and the polar cosolvent, causing change of the
substrate combination of R-enantiomer more than the other, and
hence, higher enantioselectivity was observed. Secondly, the im-
proved penetrability of cell membrane and wall led to an altered
2 4
dried over anhydrous Na SO and subjected to GC analysis. All exper-
iments were conducted in triplicate if not specified.
One unit of amidase activity was defined as the amount of enzyme
required to produce 1 μmol of (R,S)-2 per minute under the above
conditions.
2
.4. Scale-up resolution of (R,S)-2,2-dimethylcyclopropanecarboxamide
A 5-l scale reaction was performed in an 8-l stirring-tank reactor
containing 100 g of wet free cells,, 28.5 g of (R,S)-1, 250 ml of acetoni-
trile and 4750 ml of Tris–HCl buffer (50 mM, pH 8.2). The resulting
mixture was stirred at 120 rpm with the temperature maintained at
3
0 °C. After reaction for 140 min, the reaction broth was centrifuged
to remove the cells. The supernatant was treated by activated carbon
absorption (0.3%, w/v) for 30 min at 45 °C and filtration. Thereafter,
the solvent was basified to pH 12 and (S)-1 was purified via macro-
porous resin HP-1 using 80% ethanol as elution solvent. The eluent
was evaporated under reduced pressure to afford white solid. The
1
product was characterized by FT-IR spectroscopy, GC–MS, H NMR
and 13C NMR. Optical purity of the product was determined by chiral
gas chromatography and polarimetry.
(
S)-2,2-dimethylcyclopropanecarboxamide was isolated as white
20
powder; yield: 12.4 g (0.109 mol, 43.5%); [α]
D
+82.2° (c 1.0,
2
0
CH
3
OH) {lit.: [α]
D
+82° (c 1.0, CH
3
OH) [26]}, >99% ee by GC; νmax
-
−
1
+
(
(
(
KBr) (cm ) 3384, 3194, 1650, 1621; m/z (EI) 113 (M , 63%), 98
44), 96 (47), 81 (18), 72 (55), 70 (92), 55 (100), 41 (81); H NMR
CDCl , 400 MHz): δ=0.76–0.79 (q, 1H, J=4.4 Hz), 1.09–1.11(t, 1H,
3
1
Fig. 1. Effect of various cosolvents (10%, v/v) on catalytic activity and enantioselectivity
of the amidase catalyzed hydrolysis. Symbols: enzyme activity (■); enantiomeric ratio
□). Reaction conditions: 4.5 ml Tris–HCl buffer (pH 8.2), 0.5 ml of different organic
J=4.8 Hz), 1.16 (s, 3H), 1.20 (s, 3H), 1.30–1.34 (q, 1H, J=5.6 Hz);
(
13
3
C NMR (CDCl , 125 MHz): δ 174.3, 28.3, 27.0, 21.8, 20.5, 18.6.
solvent, 30 mM (R,S)-1, and 0.3 g wet cell, 30 °C, 10 min.