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Table 2 Comparison of biocatalyst efficiency in asymmetric reduction of a-chloroacetophenone
Activitya
[U mgÀ1
Sub. concn.
Time
[h]
Conv.
[%]
S/Cb
STYc
TTN of
Entry
Enzyme
]
[g LÀ1
]
ee [%]/[R/S]
[g gÀ1
]
[g LÀ1 dÀ1
]
cofactord
Ref.
1
2
3
4
5
6
7
8
LBADH
CMCR
KRED112
LsADH
KtCR
DhCR
DhCRV9
clDhCRV9
—
7.7
10
4
144
154
100
100
450
24
12
12
24
12
24
6
90
100
72
99/S
99/S
99/S
499/S
499/S
499/S
499/S
499/S
—
—
—
—
1.93
3.33
14.3
22.5e
6.9
20
—
270
216
935
5000
11 637f
49 873f
73 550f
14a
14b
14c
9
0.818
1.424
2.97
2.73
2.13
104
5.8
104
308
100
400
300
72
499
499
499
499
12b
This work
This work
This work
104
36
a
b
À1
c
d
Note: specific activity of purified enzyme. g gÀ1: gsubstrate gbiocatalyst
.
STY: space-time yield. Total turnover number per cofactor (NADPH) of
reductase. Biocatalyst amount was calculated using dried cells. NADP+ content was ca. 1.86 mmol gÀ1 DCW.
e
f
towards a-chloroacetophenone were identified and applied
in a similar protein. These results confirmed the potential
feasibility of engineering DhCR for the highly productive synthesis
of enantiopure (S)-aryl chlorohydrins.
This work was financially supported by the National Natural
Science Foundation of China (No. 21276082 & 21536004), Ministry
of Science and Technology, P. R. China (No. 2011CB710800 &
2011AA02A210), China National Special Fund for State Key Laboratory
of Bioreactor Engineering (No. 2060204) and Huo Yingdong
Education Foundation. Thanks are due to Dr Yue-Peng Shang
for the analysis of stereoselectivities of DhCR and variants.
Notes and references
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Fig. 3 Scheme for the cross-linking of recombinant E. coli for preparation
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We investigated the repeated batch operation of freeDhCRV9 and
clDhCRV9 in a 100 mL biphasic toluene/buffer system by mechanical
agitation. Each batch was carried out with 5.0 g of a-chloroaceto-
phenone in 50 mL of toluene, 10.0 g of clDhCRV9 and 1.5 equiv. of
glucose in 50 mL of KPB (pH 6.5, 100 mM) at 30 1C and 120 rpm for
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an additional 5.0 g of a-chloroacetophenone in 50 mL of toluene
and 1.0 equiv. of glucose. Only three repeated batches with 490%
conversion could be achieved with freeDhCRV9, while the use of
clDhCRV9 allowed for nine consecutive runs with 499% conversion
and 499% ee for (S)-aryl chlorohydrins (Fig. 3). The space-time yield
and TTN of the cofactor were 300 g LÀ1 dÀ1 and 78 550, respectively.
The use of clDhCRV9 in the batch mode allowed for a furthÀe1r
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À1
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In summary, key residues in the determination of the
reducing activity of short chain dehydrogenases/reductases
Chem. Commun.
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