A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity
tion of the z’ values six columns of the microtiter plate con-
Enzyme Assays
taining either a 25 mM solution of the amino acid or amine
as negative and positive reference controls (n=48) were
used. The DAB assay was carried out at the standard reac-
tion conditions. The z’ values were calculated using the fol-
lowing equation [Eq. (1)]:
The reaction mixture for tyrosine decarboxylation consisted
of 25 mM of l-tyrosine, 0.1 mM of PLP, 1.25 U/mL of l-ty-
rosine decarboxylase in citrate-phosphate buffer (43 mM
citric acid monohydrate, 114 mM Na2HPO4, pH 5.5).[27]
Meanwhile, the l-glutamic acid decarboxylation reaction
mixture contained 25 mM L-glutamic acid, 0.1 mM PLP,
1.25 U/mL of l-glutamic acid decarboxylase in pyridine-HCl
buffer (0.1M, pH 4.6).[28] The reactions were performed in
duplicate in a final volume of 100 mL by incubation for 1
hour at 378C and 1200 rpm (Thermomixer comfort, Eppen-
dorf). The reactions were stopped by the addition of 10 mL
of trichloroacetic acid (aqueous solution 100% w/v) and
centrifuged at 14000 rpm and 48C for 3 min.
For the serine decarboxylase (SDC) production, E. coli
TOP10 cells harboring the sdc truncated gene from A. thali-
ana in an expression plasmid (pBADHisA-sdcat)[29] and E.
coli TOP10 cells containing the plasmid pBADHisA were
grown in 96-well plates containing LB medium (150 mL) and
ampicilin (100 mg/mL) overnight at 378C and 150 rpm. With
the aid of a 96-pin colony replicator, cells were transferred
to deep well plates (96-well format, 2 mL volume, Greiner
Bio-One, Alphen a/d Rijn, The Netherlands) containing
1 mL of LB medium with ampicilin and l-arabinose (0.2%
w/v) and covered with a gas permeable seal film (Breath-
Seal, Greiner Bio-One). The cells were grown for 24 h at
378C and 200 rpm.
Instead, z values were estimated using the fluorescence
intensities obtained due to the decarboxylation of l-serine
catalyzed by E. coli cell-free extracts containing or not the
activity. After confirming that the l-serine did not contrib-
ute considerably to the resulting fluorescence, E. coli lysates
carrying the empty expression vector were chosen as nega-
tive controls. This decision is based on the fact that l-serine
is expected to be metabolized by other enzymes present in
the extract, such as serine hydroxymethyltransferase
(EC 2.1.2.1) or l-serine deaminase (EC 4.3.1.17), thereby
supporting that the response obtained is only due to the
presence of the decarboxylation product.
After one hour incubation, using the mean and standard
deviation of the positive (c+) and negative controls (cÀ)
(n=48), the z coefficient was calculated employing [Eq.
(2)].
The cells were collected by centrifugation at 3500 rpm
(5810R, Eppendorf) and 48C for 35 min. The supernatant
was removed and the cells were rinsed with potassium phos-
phate buffer (50 mM, pH 7.2) and centrifuged again. The su-
pernatant was discarded and the plates were stored at
À208C overnight. Cell lysates were prepared by resuspend-
ing the cell pellets in 130 mL of potassium phosphate buffer
containing 0.1 mM of PLP, 1 g/L lysozyme from egg white
and 0.1 g/L of DNAse I. After 1 h incubation at 378C, the
suspensions were centrifuged as before.
Acknowledgements
We thank Dr. Mirjam Foti and Dr. Harald J. Ruijssenaars
(TNO Quality of Life, Bioconversion Group, Delft, The
Netherlands) for providing the E. coli strain harbouring the
SDC gene from Arabidopsis thaliana. This study has been fi-
nancially supported by the Netherlands Ministry of Econom-
sic.nl) through B-Basic, a public private NWO-ACTS pro-
gramme (ACTS: Advanced Chemical Technologies for Sus-
tainability).
Protein concentration was determined by the Bradford
assay (Bio-Rad, Munich, Germany) using bovine serum al-
bumin (Bio-Rad, Munich, Germany) as standard.
For the microtiter plate reactions, aliquots of cell lysates
were transferred (90 mL) to a new microtitre plate contain-
ing 10 mL of l-serine (100 mM) dissolved in potassium phos-
phate buffer (50 mM, pH 7.2). The plates were incubated at
378C for 1 h and 200 rpm.
In the case of l-serine decarboxylation reactions, 90 mL of
cell lysates were transferred to Eppendorf tubes containing
10 mL of l-serine (250 mM) dissolved in potassium phos-
phate buffer (50 mM, pH 7.2). The reactions were per-
formed in duplicate for 1 hour at 378C and 1200 rpm and
stopped by the addition of 10 mL of trichloroacetic acid
(aqueous solution 100% w/v) and centrifuged at 14000 rpm
and 48C for 3 min.
References
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J. L. Reymond, Curr. Opin. Biotechnol. 2004, 15, 314–
322.
The formations of tyramine, GABA and ethanolamine in
the supernatant were measured using the method detailed
previously.
[2] B. Valastro, A. Dekundy, F. Tennigkeit, H. Russ, L
Franke, Patent WO2010/063486, 2010.
[3] E. M. August, D. Rajotte, Patent WO2006/055753, 2006.
[4] N. Teruyuki, K. Naoki, H. Takeshi, U. Yoichiro, U.S.
Patent 5,846,792, 1998.
Determination of z and z’ Values
To evaluate the overall performance of our decarboxylation
assay and its application in a HTS format the z and z’
values for each reaction were calculated. For the determina-
[5] X. Yibin, H. Bradford, C. Cassandra, U.S. Patent 2010/
0261668, 2010.
Adv. Synth. Catal. 2011, 353, 2369 – 2376
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