Z. Zhang / Bioorg. Med. Chem. Lett. 19 (2009) 1129–1131
1131
Figure 1. Depicted are chromatograms of the D-ES scavenging process corresponding to entry 2, 3, 5, 6, 7, and 10. The black, red, and blue lines are of the samples at the
beginning, the middle, and the end of the reaction, respectively. The retention times (min) varies from one substrate to another. The
consumed.
D-enantiomer which elutes first was
10. May, O.; Verseck, S.; Bommarius, A.; Drauz, K. Org. Process Res. Dev. 2002, 6, 452.
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Biotechnol. Progr. 1991, 7, 380.
13. Krebs, H. A. Biochem. J. 1935, 29, 1620.
14. Todone, F.; Vanoni, M. A.; Mozzarelli, A.; Bolognesi, M.; Coda, A.; Curti, B.;
Mattevi, A. Biochemistry 1997, 36, 5853.
15. Shapira, R.; Austin, G. E.; Mirra, S. S. J. Neurochem. 1998, 50, 69.
16. Sacchi, S.; Lorenzi, S.; Molla, G.; Pilone, M. S.; Rossetti, C.; Pollegioni, L. J. Biol.
Chem. 2002, 277, 27510.
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2006, 128, 10923.
using purified enzymes, and the isolation and determination of the
products formed from these enzymes would certainly help in
understanding the ES process. Nevertheless, this work uncovers a
rich realm of discoveries. The practical usefulness of this work pro-
vides a valuable target for sequencing, cloning, expression, genetic
engineering, biochemical study, biocatalytic exploration, as well as
biomedical studies, etc.
In conclusion, the whole cell culture of the A. sp. containing
multiple enzymes can be used as
the rac-AADs, affording a series of
D
-ES to remove the
D-AADs in
L
-AAD in high ee. This work pre-
18. Sacchi, S.; Rosini, E.; Molla, G.; Pilone, M. S.; Pollegioni, L. Protein Eng. Des. Sel.
sents a simple and efficient synthetic method that uses no solvents
and requires no derivatization steps. Besides being immediately
useful as a new purification method, this work opens doors to
many new possibilities in the area of biocatalysis. We anticipate
that the concept of ES can be applied to a broader range of uses
in a variety of fields.
Ongoing work is being done in getting more insight on the pro-
cess. They include mainly (1) the identification of the enzymes
responsible for the reaction in this ES system; (2) the investigation
of its mechanism and the scope of its application.
2004, 17, 517.
19. Svedendahl, M.; Hult, K.; Berglund, P. J. Am. Chem. Soc. 2005, 127, 17988.
20. Forconi, M.; Herschlag, D. J. Am. Chem. Soc. 2005, 127, 6160.
21. Note 1: The bacterium strain was identified as Aeromonas sp. and deposited in
China General Microbiological Culture Collection Center for preservation. The
deposit number is: CGMCC No. 2226.
22. Note 2: A typical experimental procedure of the bioconversion is as follows:
The bacterium A. sp. was inoculated under sterilized conditions in a 500-mL
flask containing 100 mL of growth media (yeast extract 1.0%, peptone 1.0%,
K2HPO4 0.5%, and
L-phenylalanine 0.5%, the pH was not adjusted after
sterilization) and incubated under 30 °C for 24–26 h. The culture was then
harvested, centrifuged, washed twice with ice water, and the supernatant was
decanted. The culture cake was put back into a shaking flask. The substrate
(50 mg) was put into the flask after being dissolved in dilute sodium
hydroxide. The pH was quickly adjusted to 7.3, and the total volume was
adjusted to 50 mL by tris buffer. The flask was aerated before being closed by
an air permeable stopper. The flask was then set in the shaker with a controlled
speed (200 r/min) and temperature (30 °C). The progress of the bioconversion
was monitored by regular sampling and HPLC analysis. The reaction mixture
was allowed to react for 24–48 h and extended to 53 h in some cases
depending on the conversion. For the samplings, 1 mL of reaction mixture was
removed to an Eppendorff vial and centrifuged. The cell debris was decanted,
and the supernatant was filtered. The filtrate was diluted by distilled water and
used for injection into HPLC column for analysis. The HPLC system was
equipped with a chiral column Crownpak CR (+) (Daicel, Japan). The standard
conditions of elution for rac-Phe suggested by the column supplier were
modified by adding 10% of methanol to isocratically elute all the AADs. In all
cases, the clear (or baseline) separation of the two enantiomers was achieved.
The detection wavelength was set at 200 nm. For the isolation of products, the
experiment was carried out in duplicates (maximum in parallel with 28 flasks).
At the end of transformation, the mixture was collected and centrifuged. The
cell debris was decanted, and the supernatant was loaded onto Resin column
and eluted by buffer. The elution conditions differ from one substrate to
Acknowledgments
The author expresses his acknowledgment to Professor Li-he
Zhang for his encouragement, and Professor Guo-qiang Lin for
helpful discussions. The author extends also his acknowledgment
to Ye-bin Zhang and Hong-ying Guo for technical assistance. This
work was supported by The National Natural Science Foundation
of China (Project No. 20542006).
References and notes
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another with no optimization. The fractions were also analyzed by HPLC.
AADs were collected in high purity after concentration.
L-
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