822
Y.-M. SEO et al.
100
80
60
40
20
0
fluoroalanine from rac-3-fluoroalanine. To enhance the
A
catalytic activity, a fusion protein (VHb-DAAO) of
D-amino acid oxidase (DAAO) with Vitreoscilla hemo-
globin (VHb) was used for the reaction, by which
500 mM rac-3-fluoroalanine was successfully resolved
with simple agitation. Moreover, the VHb-DAAO
reaction is applicable to the kinetic resolution reaction
of unnatural amino acids, the production of ꢀ-keto acids
from essential D-amino acids, and deracemisation of
unnatural amino acids.
0
2
4
6
8
10
Time (h)
Acknowledgment
100
80
60
40
20
0
B
This research was supported by Basic Science
Research Program through the National Research
Foundation of Korea funded by the Ministry of
Education, Science and Technology (no. 20100028158).
References
1) Goncalves LPB, Antunes OAC, Pinto GF, and Oestreicher EG,
J. Fluorine Chem., 124, 219–227 (2003).
0
2
4
6
8
10
12
14
2) Kukhar VP and Soloshonok VA, ‘‘Fluorine Containing Amino
Acids,’’ John Wiley & Sons, New York, pp. 139–220 (1995).
3) Kollonitsch J and Barash L, J. Am. Chem. Soc., 98, 5591–5593
(1976).
Time (h)
Fig. 2. Kinetic Resolution of 100 mM (A) and 500 mM (B) rac-3-
Fluoroalanine by VHb-DAAO.
4) Ohshima T, Wandey C, and Conrad D, Biotechnol. Bioeng., 34,
394–397 (1989).
A, reaction conditions: 100 mM substrate, VHb-DAAO (0.08
mg/mL), 1 mM FAD, catalase (0.05 mg/mL), and 1 mL of 100 mM
phosphate buffer (pH 8.0). B, reaction conditions: 500 mM substrate,
VHb-DAAO (0.4 mg/mL), 1 mM FAD, catalase (0.05 mg/mL), and
10 mL of 200 mM phosphate buffer (pH 8.0). Symbols: , the ee
value of (R)-3-fluoroalanine; and , the conversion of substrate.
5) Goncalves LPB, Antunes OAC, Pinto GF, and Oestreicher EG,
Tetrahedron:Asymmetry, 11, 1465–1468 (2000).
6) Hoveyda HR and Pinault JF, Org. Lett., 8, 5849–5852 (2006).
7) Gablera M, Henselb M, and Fischer L, Enzyme Microb.
Technol., 27, 605–611 (2000).
8) Caligiuri A, D’Arrigo P, Rosini E, Tessaro D, Molla G, Servi S,
and Pollegioni L, Adv. Synth. Catal., 348, 2183–2190 (2006).
9) Caligiuri A, D’Arrigo P, Gefflaut T, Molla G, Pollegioni L,
Rosini E, Rossi C, and Servi S, Biocatal. Biotrans., 24, 409–413
(2006).
for (R)-3-fluoroalanine, (S)-3-fluoroalanine, and L-Val
were observed to be 5.20, 6.81, and 9.01 min respec-
tively. The enantiomeric excess (ee) of (R)-3-fluoroala-
nine reached >99% with a conversion of 51.2%
after 10 h. Enantioselectivity was calculated to be
>100 using the equation, E ¼ ln½ð1 ꢁ cÞð1 ꢁ eesÞꢄ=
In½ð1 ꢁ cÞðl þ eesÞꢄ, formulated by Chen et al.18) Finally,
a higher concentration reaction (500 mM rac-3-fluoroa-
lanine) was carried out with 4 mg of VHb-DAAO, 1 mM
FAD, and 0.5 mg of catalase in 10 mL of 200 mM
phosphate buffer (pH 8.0) under agitation (200 rpm).
The ee of (R)-3-fluoroalanine reached >99% with a
conversion of 52.4% after 15 h (Fig. 2B). When the
wild-type DAAO reaction was subjected to similar
experimental conditions, the ee value of (R)-3-fluoroa-
lanine reached 82% with a conversion of 46% after 15 h,
suggesting that VHb-DAAO is a better catalyst than
DAAO.
10) Suwanwong Y, Kvist M, Isarankura-Na-Ayudhya C, Tansila N,
Bulow L, and Prachayasittikul V, Int. J. Biol. Sci., 2, 208–215
(2006).
11) Khang YH, Kim IW, Hah YR, Hwangbo JH, and Kang KK,
Biotechnol. Bioeng., 82, 480–488 (2003).
12) Shin JS, Yun H, Jang JW, Park I, and Kim BG, Appl. Microbiol.
Biotechnol., 61, 463–471 (2003).
13) Yun H, Hwang BY, Lee JH, and Kim BG, Appl. Environ.
Microbiol., 71, 4220–4224 (2005).
14) Kuan I, Liao R, Hsieh H, Chen K, and Yu C, J. Biosci. Bioeng.,
105, 110–115 (2008).
15) Casalin P, Pollegioni L, Curti B, and Simonetta MP, Eur. J.
Biochem., 197, 513–517 (1991).
16) Alonso J, Barredo JL, Diez B, Mellado E, Salto F, Garcia JL,
and Cortes E, Microbiology, 144, 1095–1101 (1998).
´
´
´
17) Vikartovska-Welwardova A, Michalkova E, Gemeiner P, and
Welward L, Folia Microbiol., 44, 380–384 (1999).
18) Chen CS, Fujimoto Y, Girdaukas G, and Shi CJ, J. Am. Chem.
Soc., 104, 7294–7299 (1982).
In summary, to our knowledge, this is the first study in
which the DAAO reaction was used to produce (R)-3-