Shao-Yun Chen et al.
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amplified from genomic DNA of Bacillus subtilis 168, Pichia
pastoris GS115, and Candida boidinii, respectively.
When bioconversion was complete, all reaction solutions
were processed as described above to afford the products as
oils.
Expression vectors were constructed by standard methods
and were verified by sequencing (Supporting Information,
Figures S39–S67). The vectors, which showed the highest fi-
delity, were used in transformation into expression hosts.
Engineered strains that co-expressed several enzymes were
established by co-transformation with two vectors or by
transformation with one vector containing several genes.
Recombinant E. coli strains were cultivated at 378C in
LB medium. Appropriate antibiotics were supplied, accord-
ing to resistance of the strains at the following dosages: ka-
namycin (50 mgmLÀ1), ampicillin (100 mgmLÀ1), chloromyce-
tin (34 mgmLÀ1), and streptomycin (50 mgmLÀ1). For expres-
sion on pET-30a(+), pACYCDuet-1, or pCDFDuet-1, the
cells were begun to be induced by isopropyl ß-d-1-thiogalac-
topyranoside (final concentration=0.5 mM) at 188C when
the OD600 reached approximately 0.6 (for 18–24 h). For ex-
pression on pBV220, the cells were begun to be induced by
cultivation at 428C when the OD600 reached approximately
0.6 (for 4–5 h). The cells were harvested by centrifugation
(8,000 g, 5 min, 48C), washed with 100 mM HEPES buffer
(pH 7.0), and resuspended in half the volume of this buffer.
The cell suspension was subjected to ultrasonic cell disrup-
tion to obtain the cell lysate.
(S)-2a: 1H NMR (400 MHz, DMSO-d6, 258C): d=1.181
(t, J=7.2 Hz, 3H, CH3), 2.471 (m, 2H, CH2), 3.575 (m, 2H,
CH2), 4.058 (m, 2H, CH2), 4.078 (m, 1H, CH); 13C NMR
(400 MHz, DMSO-d6): d=14.402 (CH3), 39.678 (CH2),
49.346 (CH2), 60.274 (CHOH), 67.656 (CH), 170.985
(COO); [a]2D5: À19.4 (c=1, CH3CH2OH).
(R)-2a: 1H NMR (400 MHz, DMSO-d6, 258C): d=1.181
(t, J=7.2 Hz, 3H, CH3), 2.471 (m, 2H, CH2), 3.5745 (m, 2H,
CH2), 4.058 (m, 2H, CH2), 4.078 (m, 1H, CH); 13C NMR
(400 MHz, DMSO-d6): d=14.401 (CH3), 39.675 (CH2),
49.347 (CH2), 60.272 (CHOH), 67.654 (CH), 170.975
(COO); [a]2D5: +10.2 (c=1, CH3CH2OH).
1
(S)-2b: H NMR (400 MHz, DMSO-d6, 258C): d=3.7025
(m, 2H, CH2), 4.7735 (m, 1H, CH), 7.33 (m, 5H, Ph-H);
13C NMR (400 MHz, DMSO-d6): d=50.684 (CH2Cl), 72.769
(CHOH), 126.736, 126.736, 127.827, 128.407, 128.407,
142.601 (Ph); [a]2D5: +46.2 (c=1, CH3CH2OH).
(R)-2b: 1H NMR (400 MHz, DMSO-d6, 258C): d=3.713
(m, 2H, CH2), 4.782 (m, 1H, CH), 7.341 (m, 5H, Ph-H);
13C NMR (400 MHz, DMSO-d6): d=50.680 (CH2Cl), 72.762
(CHOH), 126.737, 126.737, 127.824, 128.405, 128.405,
142.602 (Ph); [a]2D5: À42 (c=1, CH3CH2OH).
(S)-2d: 1H NMR (400 MHz, DMSO-d6, 258C): d=3.662
(m, 2H, CH2), 3.736 (s, 3H, CH3), 4.7125 (m, 1H, CH),
5.714 (d, J=4.8 Hz, 1H, OH), 6.8975 (d, J=8.4 Hz, 2H, Ph-
H), 7.314 (d, J=8 Hz, 2H, Ph-H); 13C NMR (400 MHz,
DMSO-d6): d=51.474 (CH2Cl), 56.115 (OCH3), 73.178
(CHOH), 114.515, 114.515, 128.647, 128.647, 135.378,
159.764 (Ph); [a]2D5: +26.9 (c=1, CH3CH2OH).
The protein expression was examined by SDS-PAGE. The
samples for SDS-PAGE were prepared in two steps. First,
the cell lysate was centrifuged (12,000 rpm, 5 min, 48C); the
supernatant was used to prepare a supernatant extract of
cells, and the sediment was used to prepare a sediment ex-
tract of cells after resuspension in isopycnic HEPES buffer.
Second, the extract of cells was added with loading buffer,
and followed by subsequent denaturation at 998C for
10 min.
1
(S)-2f: H NMR (400 MHz, DMSO-d6, 258C): d=3.7455
(m, 2H, CH2), 5.015 (m, 1H, CH), 6.993 (t, J=4 Hz, 1H,
CH), 7.058 (d, J=3.2 Hz, 1H, CH), 7.4335 (d, J=6 Hz, 1H,
CH). 13C NMR (400 MHz, DMSO-d6): d=51.222 (CH2Cl),
69.961 (CHOH), 125.235 (CHS), 125.98 (CH), 127.835
(CH), 147.442 (CS); [a]2D5: +28.2 (c=1, CH3CH2OH).
Biocatalysis Reaction and Product Characterization
1
(R)-3a: H NMR (400 MHz, DMSO-d6, 258C): d=1.1845
All biocatalysis reactions were carried out at 308C in
a buffer system composed of 100 mM HEPES (pH 7.0) on
a 20-mL scale in a 100-mL glass flask on a shaker (200 rpm).
The concentration of substrates (a-halo ketones) was
10 mM, the NaCN concentration was 30 mM, and the con-
centration of the co-substrates for cofactor regeneration was
100 mM or 200 mM. During incubation, 400 mL samples
were periodically removed for analysis. Promising reactions
were repeated in triplicate.
The products were analyzed as follows. Solid NaCl was
added to the samples until the solution was saturated, and
then the cells and undissolved NaCl were removed by cen-
trifugation (12,000 rpm, 1 min, 48C). The samples were ex-
tracted twice with isopycnic EtOAc, and the extracts were
centrifuged (8000 rpm, 5 min) to facilitate phase separation.
The organic phase was dried over sodium sulfate and ana-
lyzed by gas chromatography for determination of conver-
sion and ee values. In cases where HPLC analysis was re-
quired for determination of the ee value, the volume of the
organic phase was reduced as much as possible by means of
the membrane pump vacuum of a rotation evaporator, and
the residue redissolved in chromatography-grade pure etha-
nol.
(t, J=7 Hz, 3H, CH3), 2.468 (m, 2H, CH2), 2.6585 (m, 2H,
CH2), 4.063 (m, 2H, CH2), 4.134 (m, 1H, CH), 5.586 (d, J=
4.8 Hz, 1H, OH); 13C NMR (400 MHz, DMSO-d6): d=
14.372 (CH3), 25.515 (CH2), 41.454 (CH2), 60.366 (CH2),
63.773 (CH), 118.991 (CN), 170.614 (COO); [a]2D5: À20.2
(c=1, CH3CH2OH).
(S)-3a: 1H NMR (400 MHz, DMSO-d6, 258C): d=1.178
(t, J=7.2 Hz, 3H, CH3), 2.463 (m, 2H, CH2), 2.653 (m, 2H,
CH2), 4.065 (m, 2H, CH2), 4.1365 (m, 1H, CH), 5.584 (d,
J=5.6 Hz, 1H, OH); 13C NMR (400 MHz, DMSO-d6): d=
14.368 (CH3), 25.509 (CH2), 41.453 (CH2), 60.364 (CH2),
63.776 (CH), 118.99 (CN), 170.613 (COO); [a]2D5: +12.3 (c=
1, CH3CH2OH).
(S)-3b: 1H NMR (400 MHz, DMSO-d6, 258C): d=2.86
(m, 2H, CH2), 4.908 (m, 1H, CH), 5.9705 (d, J=4.4 Hz, 1H,
OH), 7.355 (m, 5H, Ph-H); 13C NMR (400 MHz, DMSO-
d6): d=27.894 (CH2), 68.426 (CHOH), 119.061 (CN),
126.114, 126.114, 127.907, 128.510, 128.510, 143.440 (Ph);
[a]2D5: +62 (c=1, CH3CH2OH).
1
(R)-3b: H NMR (400 MHz, DMSO-d6, 258C): d=2.8675
(m, 2H, CH2), 4.917 (s, 1H, CH), 5.9835 (d, J=3.6 Hz, 1H,
OH), 7.362 (m, 5H, Ph-H); 13C NMR (400 MHz, DMSO-
d6): d=28.651 (CH2), 69.176 (CHOH), 119.827 (CN),
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ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2013, 355, 3179 – 3190