flask. When the optical density of cell culture reached 0.4–0.6 at
600 nm, 0.5 mM of IPTG was added. The C-terminal His6-
tagged proteins were purified at 4 °C on a Ni-NTA agarose resin
purchased from Qiagen (Hilden, Germany). The eluted solution,
containing purified protein from Ni-NTA agarose, was dialyzed
against 100 mM sodium phosphate buffer (pH 8.0) containing
7% glycerol, 2 mM EDTA, and 5 mM 2-mercaptoethanol, and
concentrated using an Amicon ultrafiltration unit (Millipore,
USA). Glycerol was added to the purified enzyme solution (25%
glycerol) and stored at −20 °C for further studies. For the whole
cell preparation of VHb-DAAO, the cell pellet was washed twice
with 100 mM phosphate buffer (pH 8.0) after induction. Sub-
sequently glycerol was added to the whole cell solution (25%
glycerol) and stored at −70 °C for further study.
carrying only pBAD/HisA:ω-TA was similar to that of the above
mentioned procedure except for the introduction of only 50 mg
L−1 of Amp to the cell culture and the addition of only arabinose
(0.02%) to the culture media.
Acknowledgements
This study was supported by the Basic Science Research
Program (no. 20100028158) through the National Research
Foundation of Korea and the 21C Frontier Microbial Genomics
& Applications Center Program (no. 1120081800300) funded by
the Ministry of Education, Science, and Technology, Korea.
In the case of ω-TA, the coding region of ω-TAVf was
amplified and cloned into pET24ma vector and was then intro-
duced into E. coli BL21.18 The transformant of E. coli BL21
were grown in 1 L of LB broth containing 50 mg L−1 of kana-
mycin at 37 °C. When the OD600 of transformants reached
0.4–0.6, 0.5 mM of IPTG was added. After 6 h of induction, the
cells were harvested and washed twice with 50 mM phosphate
buffer (pH 8.0). After centrifugation, the cell pellet was resus-
pended in 20 mL of 50 mM phosphate buffer (pH 8.0) contain-
ing 20 μM PLP, 2 mM EDTA, 1 mM PMSF, 1 mM DTT, and
10% glycerol. Crude extracts were obtained by cell disruption
using sonication. The crude extract of the recombinant E. coli
cell containing His-tagged ω-TAVf was applied directly to Ni-
NTA agarose resin which was purchased from Qiagen (Hilden,
Germany). The enzyme was then purified as described else-
where.18 The purified enzyme was then dialyzed against 50 mM
potassium phosphate buffer (pH 7.0) containing 20 μM PLP and
concentrated with an Amicon ultrafiltration unit (Millipore,
USA). Glycerol was added to the purified enzyme solution (25%
glycerol) and stored at −20 °C for further studies. For the whole
cell preparation of ω-TA, the cell pellet was washed twice with
100 mM phosphate buffer (pH 8.0) after induction. Sub-
sequently, glycerol was added to the whole cell solution (25%
glycerol) and stored at −70 °C for further study.
Notes and references
1 S. Shin and B. G. Kim, Biotechnol. Lett., 2009, 31, 1595.
2 H. S. Bea, H. J. Park, S. H. Lee and H. Yun, Chem. Commun., 2011, 47,
5894.
3 (a) T. H. Maier, Nat. Biotechnol., 2003, 21, 422; (b) J. S. Ma, Chim.
OGGI, 2003, 21, 65.
4 (a) W. Leuchtenberger, K. Huthmacher and K. Drauz, Appl. Microbiol.
Biotechnol., 2005, 69, 1; (b) B. Seisser, R. Zinkl, K. Gruber,
F. Kaufmann, A. Hafner and W. Kroutil, Adv. Synth. Catal., 2010, 352,
731.
5 (a) A. S. Bommarius, K. Drauz, K. Günther, G. Knaup and M. Schwarm,
Tetrahedron: Asymmetry, 1997, 8, 3197; (b) H. K. Che-nault, J. Dahmer
and G. M. Whitesides, J. Am. Chem. Soc., 1989, 111, 6354;
(c) I. N. Taylor, R. C. Brown, M. Bycroft, G. King, J. A. Littlechild, M.
C. Lloyd, C. Praquin, H. S. Toogood and S. J. C. Taylor, Biochem. Soc.
Trans., 2004, 32, 290; (d) M. I. Youshko, L. M. van Langen,
R. A. Sheldon and V. K. Švedas, Tetrahedron: Asymmetry, 2004, 15,
1933.
6 (a) P. P. Taylor, D. P. Pantaleone, R. F. Senkpeil and I. G. Fotheringham,
Trends Biotechnol., 1998, 16, 412; (b) T. Li, A. B. Kootstra and I.
G. Fotheringham, Org. Process Res. Dev., 2002, 6, 533.
7 J. E. Baldwin, R. L. Dyer, S. C. Ng, A. J. Pratt and M. A. Russell, Tetra-
hedron Lett., 1987, 28, 3745.
8 D. J. Ager, T. Li, D. P. Pantaleione, R. F. Senkpeil, P. P. Taylor and
I. G. Fotheringham, J. Mol. Catal. B: Enzym., 2001, 11, 199.
9 E. Park, M. Kim and J. S. Kim, Adv. Synth. Catal., 2010, 352, 3391.
10 T. Miyazawa, H. Minowa, T. Miyamoto, K. Imagawa, R. Yana-gihara and
T. Yamada, Tetrahedron: Asymmetry, 1997, 8, 367.
11 K. Zhang, H. Li, K. M. Cho and J. C. Liao, Proc. Natl. Acad. Sci.
U. S. A., 2010, 107, 6234.
Co-expression of ω-TAwith VHb-DAAO
12 (a) O. Pamies and J. E. Backvall, Curr. Opin. Biotechnol., 2003, 14, 407;
(b) S. Servia, D. Tessaroa and G. P. Fantonib, Coord. Chem. Rev., 2008,
252, 715; (c) D. Koszelewski, B. Grischek, S. M. Glueck, W. Kroutil and
K. Faber, Chem.–Eur. J., 2011, 17, 378; (d) N. J. Turner, Curr. Opin.
Chem. Biol., 2004, 8, 114.
Initially, the coding region of the ω-TA enzyme was amplified
by PCR using the primers A1 (5′- CCGCTCGAG
ATGAACAAACCGCAAAGCTGG-3′) and A2 (5′- CCCAAG
13 I. Fotheringham, I. Archer, R. Carr, R. Speight and N. J. Turner,
Biochem. Soc. Trans., 2006, 34, 287.
CTTTTATTAGGCAACCTCGGCAAAG-3′)
from
pET24-
14 F. R. Alexandrea, D. P. Pantaleone, P. P. Taylor, I. G. Fotheringham,
D. J. Ager and N. J. Turner, Tetrahedron Lett., 2002, 43, 707.
15 Y. H. Lin, Y. F. Li, M. C. Huang and Y. C. Tsai, Biotechnol. Lett., 2004,
26, 1067.
16 Y. H. Khang, I. W. Kim, Y. R. Hah, J. H. Hwangbo and K. K. Kang, Bio-
technol. Bioeng., 2003, 82, 480.
17 Y. M. Seo, Y. H. Khang and H. Yun, Biosci., Biotechnol., Biochem.,
2011, 75, 820.
18 J. H. Seo, D. Kyung, K. Joo, J. Lee and B. G. Kim, Biotechnol. Bioeng.,
2011, 108, 253.
ma:ω-TA. The PCR product was then digested with XhoI and
HindIII, and was subsequently inserted into the pBAD/HisA
vector (Invitrogen, USA). The pBAD/HisA:ω-TA was then intro-
duced into the E. coli (BL21) containing pET24ma:VHb-
DAAO. Later these transformants were grown at 30 °C in 1L LB
broth containing 50 mg L−1 of kanamycin and 50 mg L−1 of
Amp. When the OD600 reached 0.2, IPTG (0.5 mM) and arabi-
nose (0.02%) were added to culture media. After 6 h of induc-
tion, the cells were harvested and washed twice with 50 mM
phosphate buffer (pH 8.0) and the expression level was ana-
lyzed. The single expression of ω-TA using E. coli BL21
19 D. Koszelewski, K. Tauber, K. Faber and W. Kroutil, Trends Biotechnol.,
2010, 28, 324.
20 J. S. Shin and B. G. Kim, J. Org. Chem., 2002, 67, 2848.
21 H. Yun and B. G. Kim, Biosci., Biotechnol., Biochem., 2008, 72, 3030.
This journal is © The Royal Society of Chemistry 2012
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