A high-throughput screening assay for amino acid decarboxylase activity

Article abstract of DOI:10.1002/adsc.201100386

The development of sensitive and easy-to-apply high-throughput screening methods is a common need in modern biocatalysis. With these powerful analytical tools in hands, chemists can easily assess enzyme libraries to identify either novel biocatalysts or improved mutants. Within biocatalysis, amino acid decarboxylases are gaining an increased importance, with several diverse applications ranging from the synthesis of bio-commodities to medical applications (e.g., synthesis of enzyme inhibitors at the level of L-DOPA decarboxylase). Herein, an efficient and simple analytical method for high-throughput screening of amino acid decarboxylase activity is reported. The method is valid for the discrimination of a broad range of amino acid/amine pairs such as L-tyrosine/tyramine, L-DOPA/dopamine, 5-hydroxy-L-tryptophan/ serotonin, L-histidine/histamine, L-serine/ethanolamine, L-tryptophan/ tryptamine, L-glutamic acid/GABA, and L-alanine/ethylamine. It has proven its versatility by using pure substrates, mixtures, or enzymatic reactions, both coming either from commercial enzymes or derived from cell-free (crude) extracts. The limit of detection was 13 μM for ethanolamine in the presence of 50 mM L-serine, while z′ values were in the range 0.75-0.93, indicating the suitability for high-throughput screening. Copyright

Full text of DOI:10.1002/adsc.201100386

FULL PAPERS
DOI: 10.1002/adsc.201100386
A High-Throughput Screening Assay for Amino Acid
Decarboxylase Activity
Rosario Mꢀdici,a Pablo Domꢁnguez de Marꢁa,b,c Linda G. Otten,d
and Adrie J. J. Straathofa,*
a
Bioseparation Technology Group, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC
Delft, The Netherlands
Fax : (+31)-15-278-2355; phone: (+31)-15-278-2330; e-mail: A.J.J.Straathof@tudelft.nl
AkzoNobel Chemicals BV, Process and Product Technology Department, Velperweg 76, 6800 SB Arnhem, The
b
Netherlands
c
Present address: Institute of Technical and Macromolecular Chemistry (ITMC) RWTH Aachen University, Worringerweg
1, 52074 Aachen, Germany
d
Biocatalysis and Organic Chemistry Group, Department of Biotechnology, Delft University of Technology, Julianalaan
136, 2628 BL Delft, The Netherlands
Received: May 16, 2011; Revised: July 10, 2011; Published online: August 30, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100386.
Abstract: The development of sensitive and easy-to-
apply high-throughput screening methods is
common need in modern biocatalysis. With these amine, l-glutamic acid/GABA, and l-alanine/ethyl-
powerful analytical tools in hands, chemists can amine. It has proven its versatility by using pure sub-
ACHTUNGTRENNUNG
a
AHCTUNGTRENNUNG
easily assess enzyme libraries to identify either novel strates, mixtures, or enzymatic reactions, both
biocatalysts or improved mutants. Within biocataly- coming either from commercial enzymes or derived
sis, amino acid decarboxylases are gaining an in- from cell-free (crude) extracts. The limit of detection
creased importance, with several diverse applications was 13 mM for ethanolamine in the presence of
ranging from the synthesis of bio-commodities to 50 mM L-serine, while z’ values were in the range
medical applications (e.g., synthesis of enzyme inhib- 0.75–0.93, indicating the suitability for high-through-
itors at the level of l-DOPA decarboxylase). Herein, put screening.
an efficient and simple analytical method for high-
throughput screening of amino acid decarboxylase
activity is reported. The method is valid for the dis- Keywords: amino acid decarboxylation; amino acids;
crimination of a broad range of amino acid/amine enzyme catalysis; fluorescence; high-throughput
pairs such as l-tyrosine/tyramine, l-DOPA/dop- screening
Introduction
tyrosine decarboxylases,[4] S-adenosyl-l-methionine
decarboxylase;[5] or in food industry, e.g., production
The development of sensitive and simple high- of aroma by phenylalanine decarboxylase.[6] Likewise,
throughput screening methods is a key-step in the amino acid decarboxylases are gaining increased im-
identification of novel biocatalysts, as well as in their portance in the production of bio-based commodities,
improvement towards a certain application. In this re- starting from proteins and amino acids.[7] In this re-
spect, several detection methods (e.g., colorimetric, spect, a number of processes have been recently ad-
fluorometric, etc.) have been developed and imple- dressed, involving l-aspartate-a-decarboxylase,[8] l-
mented in enzyme-based research.[1]
glutamic acid-a-decarboxylase,[9] serine decarboxy-
Amino acid decarboxylation is a key (bio)chemical lase,[10] tyrosine decarboxylase,[11] or lysine and orni-
step from which many practical uses can be derived. thine decarboxylases,[12,13] among several examples.
Applications can be found in pharmaceuticals, e.g.,
For these applications, the development of simple
drug design at the level of l-DOPA or 5-hydroxytryp- and efficient analytical methods that enable the high-
tophan decarboxylases,[2] histidine decarboxylases,[3] throughput quantification of the (decarboxylated)
Adv. Synth. Catal. 2011, 353, 2369 – 2376
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2369

Rosario Mꢀdici et al.
FULL PAPERS
amine in the presence of the amino acid precursor is
of pivotal importance. Yet, that selective discrimina-
tion is chemically challenging, and so far analytical
strategies are rather scarce.[14–18] Therefore, providing
a sensitive and simple analytical method for high-
throughput screening may clearly be an important
asset for research purposes, e.g., enabling the devel-
opment of mutants with an enhanced decarboxylase
activity within a reasonable timeframe. Herein a high-
Scheme 2. 1-Substituted thio-2-substituted isoindole for mer-
captoethanol as thiol.
In a first step, a broad range of representative
throughput screening (HTS) assay for amino acid de- amino acids and their amine counterparts was as-
carboxylase activities is provided.
sessed with the method, under HTS conditions (sam-
ples of <200 mL). To avoid leaking problems when
dispensing small volumes of the alcoholic solutions of
o-diacetylbenzene and b-mercaptoethanol using a
liquid handling workstation, a mainly aqueous mix-
Results and Discussion
The herein developed high-throughput screening ture of reagents was prepared in advance and finally
method is based on the selective fluorometric re- the sample was added. Thus, assays were conducted
sponse provided by primary amines with o-diacetyl- by dispensing 155 mL of a solution containing o-diace-
benzene and mercaptoethanol.[19] Remarkably, amino tylbenzene (0.98 mM), b-mercaptoethanol (1.16 mM)
acids display hardly any fluorescence with that ap- and sodium borate buffer (0.2M, pH 9.5), followed by
proach (Scheme 1).
2.5 mL of the sample. Every 10 min during two hours
The use of aldehydes (e.g., o-phthalaldehyde, OPA) analyses were performed in 96-well plates at excita-
for derivatizing and detecting amino acids has been tion and emission wavelength (355 and 445 nm), em-
well documented, especially for chromatographic ploying in each case the highest values of fluorescence
analysis.[20] The fluorescent species formed in the reac- observed to construct standard curves shown in
tion of OPA in presence of a thiol and amino acid has Figure 1. Since the assay reaction is slow it should be
been identified as 1-substituted thio-2-substituted iso- used with quenching of enzymatic decarboxylation. In
indole (Scheme 2).[21] Despite this well-known ap- favorable cases the assay reagent mixture (pH 9.5)
proach, the use of ketones for such purposes (e.g., o- will arrest the decarboxylation.
diacetylbenzene) has been testimonial, and thus a de-
As depicted (Figure 1), the HTS method turned out
tailed mechanistic path for that reaction remains un- to be outstanding for discriminating an ample number
known.[20] Importantly, whereas aldehydes cannot dis- of amino acid/amine pairs, with the exception of
criminate between amines and their amino acid pre- amino acids harboring a second amine group. Quite
cursors,[20] diketones display a markedly different re- remarkably, highly relevant substrates for medicinal
sponse between amines and amino acids (see below). purposes, like l-glutamic acid/g-aminobutyric acid
Scheme 1. Schematic representation of the concept of the high-throughput screening assay reported in this work.
Figure 1. Fluorescence emission curves obtained employing the proposed method, under high-throughput-screening condi-
tions, for different amine/amino acid pairs. The formation of the fluorescent complexes were recorded every 10 min during
120 min at lexc =355 nm and lem =445 nm. The curves were plotted using the highest values achieved at the following incuba-
tion time: A l-tyrosine/tyramine: 60 min, B l-DOPA/dopamine: 100 min, C 5-hydroxy-l-tryptophan/serotonin: 20 min, D l-
histidine/histamine: 40 min, E l-serine/ethanolamine: 70 min, F l-tryptophan/tryptamine: 60 min, G l-glutamic acid/GABA:
90 min, H l-alanine/ethylamine: 80 min, I l-ornithine/putrescine: 40 min. AU stands for arbitrary units. The data shown are
representative of experiments performed in quadruple and are presented as meanÆstandard deviation.
2370
asc.wiley-vch.de
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2369 – 2376

A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity
Adv. Synth. Catal. 2011, 353, 2369 – 2376
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2371

Rosario Mꢀdici et al.
FULL PAPERS
Figure 2. Time course of enzymatic decarboxylation reactions catalyzed by different amino acid decarboxylases. Reaction
mixtures contained: (left) 25 mM l-serine in aqueous potassium phosphate buffer (50 mM, pH 7.2), 0.1 mM PLP; (middle)
25 mM l-tyrosine in aqueous citrate-phosphate buffer (43 mM citric acid monohydrate, 114 mM Na2HPO4, pH 5.5), 0.1 mM
PLP and 1.25 U/mL of l-tyrosine decarboxylase; (right) 25 mM l-glutamic acid in aqueous pyridine-HCl buffer (0.1M,
pH 4.6), 0.1 mM PLP and 1.25 U/mL of l-glutamic acid decarboxylase. The formation of the product of decarboxylation was
determined using the fluorescent method described. These experiments were performed in duplicate and represented as
mean Æstandard deviation.
(GABA), l-DOPA/dopamine, or 5-hydroxy-l-trypto- formation). No substantial sources of variability (such
phan/serotonin displayed excellent discriminating re- as evaporation or trends in the response within one
sults, broadening the scope of the method to very im- column or row) were produced in the conditions as-
portant applications like clinical studies, drug design, sayed. The same trends of results were obtained for
or development of enzyme inhibitors.
the decarboxylation of l-serine carried out by E. coli
Once the method was evaluated over a wide range lysates, as example of real samples (Figure S2 in the
of amino acid/amine pairs, the approach was assessed Supporting Information).
using real samples from some enzymatic decarboxyla-
To further assess the robustness of the fluorometric
tions catalyzed by decarboxylases for serine, glutamic method, values of z (for real samples) and zꢀ (for ref-
acid and tyrosine at different conditions (cell-free or erence compounds) of some amino acid/amine pairs
crude acetone Escherichia coli extracts, or whole-cell were determined.[24–26] These parameters provide a
dried Streptococcus faecalis cells) (Figure 2). In Figure useful indication of the performance of the method.
2 (middle) and (right) – at suboptimal conditions – In a nutshell, the z factor considers the dynamic
full conversion to amine was not achieved. To provide range of the assay, and data variation associated with
a further independent confirmation of the extent of the sample and reference measurements. Thus, z pro-
reaction, HPLC (Marfeyꢃs reagent derivatization[22]
)
vides the suitability of an assay for HTS. Values of z
was conducted. However, this did not provide quanti- ranging 0.5–1.0 indicate an excellent assay, showing a
tative results for both substrate and product, render- high dynamic range (difference between the means of
ing heavily tailing peaks. In any case, results were the sample signal and the reference or negative con-
qualitatively comparable in both cases. For high- trol signal) and small standard deviations (small data
throughput screening (e.g., for identification of active variability). Conversely, the zꢀ factor is an intrinsic pa-
mutants), the fluorescence approach may be accepta- rameter giving information about the quality of the
ble.
assay itself, without using real samples. The zꢀ value is
The limit of detection of the fluorometric method calculated using positive and negative reference con-
was calculated to be 13 mM for ethanolamine in the trols, and it is generally employed for the evaluation
presence of 50 mM L-serine. In addition, fluorescence of the quality of the assay, development and optimiza-
emission values obtained were employed to assess the tion[24] (see Experimental Section for an extended dis-
spatial homogeneity of the samples, in search of pat- cussion on the z and zꢀ methodology). Calculated zꢀ
terns of drift or edge effects[23] using the pair l-gluta- values for several amino acid/amine pairs led to
mic acid/GABA as example. Therefore, the fluores- values in the range 0.5–1.0, supporting the robustness
cence emission was plotted against well number and of the method for a wide number of substrates
ordered either by row first, then by column (Figure (Table 1). Table 2 provides some underlying data.
S1A in the Supporting Information), or by column
The development of assays in a high-throughput
first, then by row (Figure S1B in the Supporting In- configuration has gained increasing attention since
2372
asc.wiley-vch.de
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2369 – 2376

A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity
Table 1. Values of zꢀ indicating the performance of the
assay.
Conclusions
In this work, an analytical method for the high-
throughput screening of amino acid/amine pairs has
been reported, based on the different response to-
wards o-diacetylbenzene and mercaptoethanol. Re-
markably, the approach is valid for a very broad
range of important compounds like dopamine, seroto-
nin, and GABA, and therefore it is expected that the
method will be useful for many clinical applications,
for biocatalytic strategies involving decarboxylases, as
well as many other innovative applications, e.g., for
Amino acid/Amine
zꢀ
l-tyrosine/tyramine
l-glutamate/GABA
l-histidine/histamine
l-serine/ethanolamine
l-DOPA/dopamine
0.91
0.89
0.88
0.93
0.92
0.91
5-hydroxy-l-tryptophan/serotonin
they provide the starting point for the identification medical research purposes or in pharmaceutical
or selection of new enzymes from libraries by using chemistry.
microbial diversity or protein engineering methods. In
a directed evolution scenario, a high degree of accura-
cy and sensitivity in the assay is critical for the iden- Experimental Section
tification of the enzyme variant that shows the higher
catalytic activity. Hence, the response variation needs Materials
to be evaluated not only in the two extremes of the
Amino acids and the respective amines were purchased
activity range but also at different substrate/product
from Sigma–Aldrich (Schnelldorf, Germany) and used with-
concentrations. For that purpose, substrate-product
out further purification [l-serine, monoethanolamine, 5-hy-
mixtures simulating the reaction progress of an enzy-
matic transformation were prepared and subjected to
analysis with the herein presented fluorometric
method (Figure 3).
droxytryptophan, serotonin hydrochloride, 3,4-dihydroxy-l-
phenylalanine (l-DOPA), dopamine hydrochloride, l-ala-
nine, ethylamine, l-tyrosine, tyramine hydrochloride, l-glu-
tamic acid, g-aminobutyric acid (GABA), l-histidine, hist-
AHCTUNGTREGaNNUN mine dihydrochloride, l-tryptophan, tryptamine hydrochlo-
The results obtained were employed to calculate z’
values and these were plotted vs. substrate conversion
for each pair of amino acid/amine (Figure S3 in the
Supporting Information). Values of z’ were in the
range of 0.75–0.93 even at 10% substrate conversions,
clearly showing that the assay is sufficiently sensitive
to identify a hit. Moreover, the performance of the
assay was tested in an entire high-throughput screen-
ing procedure (96-well plate format) to measure the
serine decarboxylase activity present in E. coli cell ly-
sates. The ethanolamine concentrations obtained in
each well after one hour of incubation were used to
determine the z value at the conditions assayed. A
value z=0.70 was obtained, which confirms the suita-
bility of the assay for future directed evolution experi-
ments.
ride, l-ornithine hydrochloride, putrescine dihydrochloride].
1,2-Diacetylbenzene (DAB), pyridoxal 5-phosphate hydrate
(PLP) were supplied by Sigma–Aldrich as well. 2-Mercap-
toethanol was obtained from Merck (Darmstadt, Germany).
The enzymes l-tyrosine decarboxylase (whole dried Strep-
tococcus faecalis cells, 1.4 U/mg) and l-glutamic acid decar-
boxylase from E. coli (crude acetone powder, 0.73 U/mg),
DNAse-I from bovine pancreas (569 kU/mg of solid) and ly-
sozyme from egg white (55100 U/mg of solid) were also
from Sigma–Aldrich.
Assay Conditions
All the liquid transfers were performed using a JANUS Au-
tomated Workstation equipped with 8-needle Varispam and
96-tip MDT arms (Perkin–Elmer, Groningen, The Nether-
lands). The assays were carried out dispensing in each well
155 mL of solution composed of 150 mL of sodium borate
buffer (0.2M pH 9.5), 2.5 mL of diacetylbenzene solution
(10 g/L in methanol, 61 mM) and 2.5 mL b-mercaptoethanol
solution (5.7 g/L in ethanol, 72 mM), followed by the addi-
tion of 2.5 mL of sample.
Table 2. Typical standard deviation (SD) values in microtiter plate assays. Values at 25 mM l-serine were used for calculating
z’.
l-Serine [mM]
Cell-free extract
Mean ÆSD [mM]
n
10
10
10
5
E. coli TOP10 pBADHisA
0.39Æ0.05
6.50Æ0.56
6.52Æ0.58
3.20Æ0.47
48
48
80
80
E. coli TOP10 pBADHisASDCat
E. coli TOP10 pBADHisASDCat
E. coli TOP10 pBADHisASDCat
Adv. Synth. Catal. 2011, 353, 2369 – 2376
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2373

Rosario Mꢀdici et al.
FULL PAPERS
Figure 3. Simulated reaction progress and standard curves. A l-tyrosine/tyramine, B l-glutamic acid/GABA, C l-histidine/
histamine, D l-serine/ethanolamine, E l-DOPA/dopamine, F 5-hydroxy-l-tryptophan/serotonin. The data shown are repre-
sentative of experiments performed in quadruple and are presented as mean ÆSD. AU stands for arbitrary units.
The fluorescence measurements were performed in 96
well-plates (Greiner Bio-One, Alphen a/d Rijn, The Nether-
lands) with excitation and emission wavelengths of 355 and
445 nm, respectively. The formation of the fluorescent com-
plexes was followed recording the fluorescence intensities
every 10 min during two hours using a Bio Tek reader (Si-
nergy 2, Bio Tek Instruments, Inc., Winoosky, VT). Standard
curves of the amino acids and their decarboxylation prod-
ucts and different substrate-product mixtures (simulating
the reaction progress of an enzymatic transformation)
curves were performed in a concentration range of 0–25 mM
in quadruple. The information obtained from the three men-
tioned curves was used to build Figure 1 Figure 2 and in the
Supporting Information, Figure S3, where the contribution
of each component of the mixture to the resulting fluores-
cence emission is indicated.
2374
asc.wiley-vch.de
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2369 – 2376

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-
ic Affairs and the B-Basic partner organizations (www.b-ba-
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
[1] a) J. L. Reymond, P. Babiak, Adv. Biochem. Eng./Bio-
technol. 2007, 105, 31–58; b) J. L. Reymond, Food Tech-
nol. Biotechnol. 2004, 42, 265–269; c) J. P. Goddard,
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
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2375

Rosario Mꢀdici et al.
FULL PAPERS
[6] H. J. Klee, D. Tieman, Patent WO2005/035752, 2005.
[7] E. L. Scott, F. Peter, J. P. M. Sanders, Appl. Microbiol.
Biotechnol. 2007, 75, 751–762.
[8] P. M. Kçnst, M. C. R. Franssen, E. L. Scott, J. P. M.
Sanders, Green Chem. 2009, 11, 1646–1652.
[9] T. M. Lammens, D. De Biase, M. C. R. Franssen, E. L.
Scott, J. P. M. Sanders, Green Chem. 2009, 11, 1562–
1567.
[19] M. Roth, Anal. Chem. 1971, 43, 880–882.
[20] P. Zuman, Chem. Rev. 2004, 104, 3217–3237.
[21] J. F. Stobaugh, A. J. Repta, L. A. Sternson, K. W.
Garren, Anal. Biochem. 1983, 135, 495–504.
[22] K. K. Ngim, J. Zynger, B. Downey, J. Chromatogr. Sci.
2007, 45, 126–130.
[23] B. Eastwood, S. Iturria, P. Iversen, C. Montrose, R.
Moore, Assay Guidance Manual, Version 6, 2009, NIH
Chemical Genomics Center (NCGC) from internet
http://assay.nih.gov/assay/index.php/Table_of_Contents,
accessed on April 2011.
[10] P. Soucaille, R. Figge, U.S. Patent 2009/0325245, 2009.
[11] J. Achkar, A. Ferrꢄndez, T. Sonke, M. G. Wubbolts,
U.S. Patent 2010/0068775, 2010.
[12] S. Verseck, H. Hꢅger, A. Karau, L. Eggeling, A. Sahm,
[24] J. H. Zhang, T. D. Y Chung, K. R. Oldenburg, J.
Biomol. Screening 1999, 4, 67–73.
[25] J. Inglese, C. E. Shamu, R. K. Guy, Nat. Chem. Biol.
Patent WO2008/092720, 2008.
[13] K. Eppelmann, P. M. M. Nossin, S. M. Kremer, M. G.
Wubbolts, Patent WO2006/005604, 2006.
2007, 3, 438–441.
[14] Y. Lin, C. Chen, C. Wang, F. Pu, J. Ren, X. Qu, Chem.
[26] J. Inglese, R. L. Johnson, A. Simeonov, M. Xia, W.
Zheng, C. P. Austin, D. S. Auld, Nat. Chem. Biol. 2007,
3, 466–479.
[27] T. Børrensen, N. K. Klausen, L. M. Larsen, H. Søren-
sen, Biochim. Biophys. Acta 1989, 993, 108–115.
[28] M. L. Fonda, Biochemistry 1972, 11, 1304–1309.
[29] D. Rontein, D. Rhodes, A. D. Hanson, Plant Cell Physi-
ol. 2003, 44, 1185–1191.
Commun. 2011, 47, 1181–1183.
[15] A. Hennig, H. Bakirci, W. M. Nau, Nat. Methods. 2007,
4, 629–632.
[16] D. M. Bailey, A. Hennig, V. D. Uzunova, W. M. Nau,
Chem. Eur. J. 2008, 14, 6069–6077.
[17] A. P. H. Phan, T. T. Noo, H. M. Lenhoff, Anal. Bio-
chem. 1982, 120, 193–207.
[18] R. M. Rosemberg, R. M. Herreid, G. J. Piazza, M. H.
OꢃLeary, Anal. Biochem. 1989, 181, 59–65.
2376
asc.wiley-vch.de
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2369 – 2376