Paper
Green Chemistry
for 12 h in 5 ml of LB medium containing 80 µg ml−1 of ampi- tion products in the supernatant were analyzed by TLC and
cillin. The grown cells (5 ml) were then transferred into 500 ml HPLC. When a new spot or new peaks were detected by TLC
of LB medium containing 80 µg ml−1 of ampicillin and 1 mM or HPLC, the product was analyzed by MS. The screening
IPTG, and cultivated at 37 °C for 24 h to express the mutated was performed using L-phenylalanine dehydrogenase from
pkDAO gene. The cells were harvested by centrifugation Bacillus sphaericus,26 L-leucine dehydrogenase from Bacillus
(5000g, 15 min), and washed with 10 mM potassium phos- sphaericus,27 L-amino acid oxidase from Crotalus atrox (CaLAO),
phate buffer (KPB), pH 8.0. After the cells (12 g of wet weight) D-amino acid oxidase from pig kidney (pkDAO), and pkDAO
were disrupted by sonication, the cell debris was discarded by Y228L/R283G (mutant pkDAO).14 TLC analysis was performed
centrifugation (20 000g, 30 min, 4 °C) and the enzyme was pur- using a solvent of hexane/ethyl acetate (1 : 1) or butanol/ethyl-
ified from the supernatant solution. The precipitates formed acetate/water (3 : 1 : 1). The reaction products containing amino
by using 20–35% saturated ammonium sulfate were dissolved groups were analyzed by the ninhydrin method on a TLC plate.
in 10 mM KPB, pH 8.0, containing 0.1% 2-mercaptoethanol
(2ME) and dialyzed against the same buffer. The dialyzed
enzyme solution was then applied to a DEAE-Toyopearl
column (10 cm × 2.8 cm diameter) equilibrated with 10 mM
Acknowledgements
KPB, pH 8.0, containing 0.1% 2-ME and eluted by using a We thank Dr. Kazunori Yamamoto, Prof. Yasumasa Kuwahara,
linear gradient of NaCl (0 to 0.15 M, 150 mL each). and Dr. Hidenobu Komeda for comments and advice concern-
Ammonium sulfate was added to the active fractions up to ing this work. The authors appreciate Dr. Daisuke Matsui and
20% saturation, then the solution was applied to a Butyl- Prof. Kimiyasu Isobe for discussions and critical reading of the
Toyopearl 650 M column (3.5 cm × 2.8 cm) equilibrated with manuscript. This work was supported by the Asano Active
10 mM KPB, pH 8.0, containing ammonium sulfate (20% sat- Enzyme Molecule Project from the Exploratory Research for
uration). The adsorbed enzyme was eluted by using a linear Advanced Technology (ERATO) of the Japan Science and
gradient of ammonium sulfate (20–0% saturation, 100 mL Technology Agency (JST).
each) in 10 mM KPB, pH 8.0. The active fractions were col-
lected and dialyzed against 10 mM KPB, pH 8.0, containing
0.1% 2-ME.
References
Nitrilase AY487533
1 A. Strecker, Justus Liebigs Ann. Chem., 1850, 75, 27–45.
E. coli BL21 (DE3) cells harboring pET15b-nitrilase were incu-
bated at 37 °C for 12 h in 5 ml LB medium containing 80 µg
ml−1 ampicillin. The grown cells (5 ml) were then transferred
into 500 ml LB medium containing 80 µg ml−1 ampicillin and
cultivated at 37 °C for 12 h. Then, 0.5 mM IPTG was added,
and further incubated at 30 °C for 12 h. The cells (23 g wet
weight) were harvested by centrifugation (5000g, 15 min), and
washed with 20 mM KPB, pH 8.0. Then, the cells were dis-
rupted by sonication, and the cell debris was removed by cen-
trifugation (20 000g, 30 min, 4 °C). The supernatant was
applied to a DEAE-Toyopearl column (10 cm × 2.8 cm dia-
meter) equilibrated with 10 mM KPB, pH 8.0, containing 0.1%
2-ME, and the adsorbed enzyme was eluted by using a linear
gradient of NaCl (0–0.5 M, 150 ml each). The active fractions
were collected and dialyzed against 10 mM KPB, pH 8.0, con-
taining 0.1% 2-ME. The dialyzed enzyme solution was concen-
trated by ultrafiltration until approximately 100 U ml−1 and
used for experiments.
2 D. Enders and J. P. Shilvock, Chem. Soc. Rev., 2000, 29, 359–
373.
3 D. B. Ushakov, K. Gilmore, D. Kopetzki, D. T. McQuade and
P. H. Seeberger, Angew. Chem., Int. Ed., 2014, 53, 557–561.
4 Y. Asano, A. Yamada, Y. Kato, K. Yamaguchi, Y. Hibino,
K. Hirai and K. Kondo, J. Org. Chem., 1990, 55, 5567–5571.
5 Y. Asano, Y. Kato, C. Levy, P. Baker and D. Rice, Biocatal.
Biotransform., 2004, 22, 131–138.
6 J. Qiu, E. Su, W. Wang and D. Wei, Tetrahedron Lett., 2014,
55, 1448–1451.
7 T. C. Bhalla, A. Miura, A. Wakamoto, Y. Ohba and
K. Furuhashi, Appl. Microbiol. Biotechnol., 1992, 37, 184–
190.
8 Y. Asano and S. Yamaguchi, J. Am. Chem. Soc., 2005, 127,
7696–7697.
9 K. Yasukawa, R. Hasemi and Y. Asano, Adv. Synth. Catal.,
2011, 353, 2328–2332.
10 R. Metzner, S. Okazaki, Y. Asano and H. Gröger,
ChemCatChem, 2014, 6, 3105–3109.
11 M. Dadashipour and Y. Asano, ACS Catal., 2011, 1, 1121–
1149.
Screening the synthesis of primary α-aminonitriles from
α-amino acids or primary amines
The production of primary α-aminonitriles by using enzymes 12 M. Dadashipour, Y. Ishida, K. Yamamoto and Y. Asano,
was performed by the incubation of 10 mM α-amino acid or Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 10605–10610.
primary α-amines with 10–200 mM KCN in the presence of 13 V. Kohler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell
amino acid oxidases or amine oxidases at pH 6–10 in a final
and N. J. Turner, Angew. Chem., Int. Ed., 2010, 49, 2182–
volume of 1.0 ml. The reactions were performed at 30 °C for
2184.
1 h and stopped by addition of 0.2 ml 2 M HClO4. The result- 14 K. Yasukawa, S. Nakano and Y. Asano, Angew. Chem., Int.
ing precipitates were removed by centrifugation and the reac-
Ed., 2014, 53, 4428–4431.
Green Chem.
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