ChemCatChem
10.1002/cctc.201801254
FULL PAPER
E. coli BL21 (DE3) cells harboring pET15b-nitrilase AY487533
W186A were cultivated under the same conditions as cells harboring
pET15b-nitrilase AY487533. The cells cultivated at 37 °C for 6 h in 500
mL of LB medium were harvested by centrifugation and washed with 20
mM KPB (pH 8.0). Then, the cells were disrupted by sonication, and the
supernatant solution was used for the synthesis of amides.
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Effects of pH and temperature on nitrilase AY487533 activity
The nitrilase activity was assayed under standard assay conditions,
except that the reaction pH was varied between pH 5.0 and 11.5 using
Citrate buffer (pH 5.0–6.0), KPB (pH 6.0–8.0), Tris-HCl (pH 8.0–9.5),
[
3]
3 4 4
CH COONH -NH OH (pH 8.0-10.5), and glycine-KOH (pH 9.5–11.5).
Effect of temperature on nitrilase activity was assayed under standard
assay conditions, except that the temperature was varied between 10
o
and 80 C.
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a) K. Yasukawa, R. Hasemi and Y. Asano, Adv. Synth. Catal. 2011, 353
Effects of pH and temperature on nitrilase AY487533 stability
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The effect of pH on enzyme stability was analyzed between pH 5.0 and
o
1
1.5 by incubation at 30 C for 30 min without the substrate using the
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same buffers as described above. The effect of temperature on enzyme
o
stability was assayed by incubation at 10-80 C with 100 mM KPB (pH
[6]
8
.0), for 30 min without the substrate. The enzyme activity was assayed
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under standard assay conditions.
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One-pot synthesis of (R)-2PG from benzaldehyde and KCN by a
combination of Strecker synthesis and nitrilase AY487533
The synthesis of (R)-2PG from benzaldehyde and KCN by one-pot
chemo-enzymatic reaction using Strecker synthesis and nitrilase
AY487533 was performed as follows. The reaction mixture (0.5 mL)
containing 50 mM benzaldehyde, 150 mM KCN and crude enzyme
solution of nitrilase AY487533 was incubated at 20 C for 60 min with 500
mM CH
addition of 0.1 mL of 2 M HClO
centrifugation was discarded. The reaction products in the supernatant
were analyzed by HPLC with Crownpack CR-I (+). The same reaction
conditions were also used for synthesis of 4-hydroxy-2PG from 4-
hydroxy-benzaldehyde and 4-fluoro-2PG from 4-fluoro-benzaldehyde.
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o
3
COONH
4
-NH
4
OH buffer, pH 8.0. The reaction was stopped by
, and precipitate formed upon
4
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2
2
One-pot synthesis of (R)-2PGNH from benzaldehyde and KCN
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2
The synthesis of (R)-2PGNH from benzaldehyde and KCN by one-pot
chemoenzymatic reaction was performed under the same reaction
conditions as the synthesis of (R)-2PG by using crude enzyme solution of
nitrilase AY487533 W186A. The reaction products were analyzed using
HPLC as described above.
[18] T. C. Bhalla, A. Miura, A. Wakamoto, Y. Ohba, K. Furuhashi, Appl.
Acknowledgements
We thank Dr. Kazunori Yamamoto, Dr. Daisuke Matsui, Dr.
Kazuyuki Yasukawa, Prof. Yasumasa Kuwahara, and Prof.
Hidenobu Komeda for their advice and comments on this work.
The authors are thankful to Prof. Kimiyasu Isobe for his
discussions and critical reading of the manuscript. This work
was supported by a grant from the Exploratory Research for
Advanced Technology (ERATO), Asano Active Enzyme
Molecule Project from the Japan Science and Technology
Agency (JST) (Grant Number JPMJER1102). This research was
also supported in part by a grant-in-aid for Scientific Research S
from The Japan Society for Promotion of Sciences (No.
17H06169) to Y. Asano.
Keywords: nitrile, amino acid, Strecker synthesis, nitrilase
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