Biocatalysed synthesis of chiral amines: continuous colorimetric assays for mining amine-transaminases

Article abstract of DOI:10.1039/d0cy02070b

In the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral amines, we have developed new continuous assays for the screening of amine-transaminase collections. These assays are based on the use of hypotaurine as an irreversible amine donor. This β-aminosulfinic acid is converted upon transamination into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily detected by spectrophotometry using Ellman's reagent. Two complementary assays were developed based on this titration method. Firstly, a direct assay allowed detection of various transaminases able to use hypotaurine as an amino donor. In a second coupled assay,l-alanine is used as a generic donor substrate of amine-transaminases and is regenerated using an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a variety of valuable new biocatalysts for use in synthetic processes.

Full text of DOI:10.1039/d0cy02070b

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Page 1 of 9
CP al et aa lsy es i ds oS c ni eo nt c ae d &j uTs et c mh na or gl oi ng sy
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DOI: 10.1039/D0CY02070B
ARTICLE
Biocatalysed Synthesis of Chiral Amines: Continuous Colorimetric
Assays for Mining Amine-Transaminases
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
b
Léa Gourbeyre, Egon Heuson, Franck Charmantray, Virgil Hélaine, Adrien Debard, Jean-Louis
b
b
a
Petit, Véronique de Berardinis, and Thierry Gefflaut *
DOI: 10.1039/x0xx00000x
In the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral
amines, we have developed new continuous assays for the screening of amine-transaminases collections. These assays are
based on the use of hypotaurine as irreversible amino donor. This β-aminosulfinic acid is converted upon transamination
into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily
detected by spectrophotometry using Ellman’s reagent. Two complementary assays were developed based on this
titration method. Firstly, a direct assay allowed detecting various transaminases able to use hypotaurine as amino donor.
In a second coupled assay, l-alanine is used as generic donor substrate of amine-transaminases and is regenerated using
an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated
through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a
variety of valuable new biocatalysts for use in synthetic processes.
and efficient solution is to use cheap isopropylamine (IPA) as
amine donor since acetone, which is formed from IPA upon
transamination, can be easily removed from the reaction
Introduction
Transaminases (TA, E.C. 2.6.1.X) which offer a highly
stereoselective access to chiral amino pharmaceuticals and
bioactive compounds have gained considerable interest in the
past two decades.1 TA are pyridoxal phosphate (PLP)-
dependent enzymes catalyzing the reversible transfer of the
amino group from an amine donor substrate onto a carbonyle
acceptor substrate. TA belong to fold-types I and IV of the
large family of enzymes using PLP for catalyzing a variety of
chemical reactions.4 The TA family is further divided into 6
classes based on sequence homology and related substrate
spectra. Furthermore, TA are classically divided into 2 groups:
α-TA which are exclusively active with α-amino and α-keto
acids and amine-TA (also called ω-TA) which are able to accept
other carbonyle and amino compounds as substrates. Most
amine-TA belong to class III (fold-type I), although some of
them have been found in class IV (fold-type IV) displaying an
9
-11
medium.
amine-TA. An alternative frequently used method is the
addition of an auxiliary enzyme to catalyse
thermodynamically favored transformation of the carbonyle
Unfortunately, IPA is not a common substrate for
a
-3
1
2-14
co-product formed from the amine donor substrate.
In
some cases, a spontaneous transformation of the co-product
formed from a smart amine donor can afford the needed
equilibrium shift. This includes cyclisation of amino or amido
carbonyle derivatives formed from diamino or dicarbonyle
-5
1
5-22
23-24
compounds,
or decomposition of β-keto acids.
Following this last approach, we used L-cysteine sulfinic acid (L-
CSA) as irreversible amine donor in AspTA-catalyzed syntheses
2
5-26
of a variety of glutamic acid analogues.
L-CSA is converted
upon transamination into an unstable β-keto sulfinic acid
which spontaneously releases sulfur dioxide. In addition to the
equilibrium shift, this chemical transformation also affords an
6
unusual stereoselectivity ((R)-amine-TA). The use of TA for
2
efficient way of measuring enzyme activity. Indeed, SO is
chiral amine synthesis brings many benefits including broad
application scope, high stereoselectivity and no need for
auxiliary cofactor recycling. However, a major drawback of
transamination lies in its reversible nature and therefore, an
hydrated into sulfite ions and easily titrated by colorimetry
using Ellman’s reagent (5,5’-dithiobis-(2-nitro-benzoic acid),
2
7-28
DTNB).
Therefore this methodology was used to develop
highly efficient high throughput screening assays for exploring
7
-8
equilibrium-shift strategy is almost always needed. A simple
2
9
α-TA collections.
Regarding the widespread application of amine-TA, the
design of continuous and high throughput screening assays
suitable for this group of enzymes is of utmost importance for
identifying new biocatalysts in large collections of wild-type or
a. Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-
Ferrand, France. E-mail: Thierry.gefflaut@uca.fr
Génomique métabolique, Genoscope, Institut François Jacob, CEA, CNRS,
b.
Université d’Evry, Université Paris-Saclay, 91057 Evry, France.
Electronic Supplementary Information (ESI) available: Experimental details, mutant enzymes.
enzyme kinetics, Enzyme library data (reference set, primers used for cloning,
sequences comparison). See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/D0CY02070B
ARTICLE
O
Direct assay
Coupled assay
R1
R2
NH2
acceptor
SO H
2
H N
2
HO C
SO2
2
HPT
Ala
HPT-TA
TA
O
+
O
SO H
HPT
2
HO C
O
2
unstable
NH2
Pyr
SO2
R1
R2
chiral amine
Figure 1. Principle of direct and coupled screening assays.
Therefore, various methods have been reported over the last However, this last screening method was not yet reported in a
years, and can be divided into two categories. The first one continuous version.
corresponds to the direct detection of one of the Herein, we report the design and validation of new
transamination products or of a spontaneously formed colorimetric and continuous assays based on the use of
derivative. The second category involves a coupled reaction hypotaurine (HPT) as irreversible amino donor and on specific
usually catalysed by an auxiliary enzyme. Hence, colorimetric titration of sulfite ions produced from HPT upon
acetophenone or acetonaphtone formed from the transamination. Two complementary assays were developed
corresponding aryl-alkylamines can be directly titrated by and used on cell lysates for the screening of a large collection
3
0-31
spectrophotometry or fluorometry,
whereas o- of putative amine-TA from microbial diversity.
xylylenediamine or p-nitro-phenylethylamine used as amino
donor results in the formation of polymerized colored
derivatives.1
7,32
Results and Discussion
Despite their simplicity, these methods are
however limited to enzymes able to use these particular amine
donors and formation of precipitated polymers can impair the
development of continuous assays. Alternatively, several
coupled-assays are based on auxiliary redox reactions. For
example, pyruvic acid (Pyr) formed from alanine (Ala), a
preferred substrate for most known amine-TA, was reduced to
lactic acid and a pH indicator was used to monitor consecutive
pH decrease.33 Alternatively, L- or D-Ala produced from Pyr,
was oxidised using auxiliary L- or D-amino acid oxidases (AAO)
As shown in figure 1, two assays based on sulfite titration were
developed. In the direct assay, HPT is used as unique amino donor
substrate and leads upon transamination to the release of sulfur
dioxide, itself instantaneously hydrated into sulfite ions which react
with DTNB to give a colored thiolate anion (λ = 412 nm, ε = 14150
-1
-1 27
M
cm ). In the coupled assay, Ala, is used as standard amine
donor and is regenerated via an auxiliary transamination catalysed
by an HPT-TA. This non-limiting coupled reaction thus allows the
activity measurement through sulfite production and can afford an
equilibrium shift.
2 2 2
converting O into H O which was itself used to oxidise a
chromogenic substrate using horse radish peroxidase as
3
4
secondary auxiliary enzyme. However, this method was
hindered by the limited availability of L-AAO for large scale
Direct assay performance and validation.
In order to develop the direct assay, we chose the transamination
between HPT and Pyr as a model reaction and ω-TA from
Caulobacter crescentus (Uniprot ID Q9A3Q9) as a model enzyme.
This amine-TA was previously shown to be active with β-alanine,
3
5
screening processes. In order to circumvent this limitation,
glyoxylic acid was used instead of Pyr as acceptor and glycine
oxidase was used as auxiliary redox enzyme for producing H
2
O
2
from achiral glycine.3 In another study, addition of an alanine-
6
3
8
the carboxylic analogue of HPT. Therefore Q9A3Q9 was cloned
and over-expressed in Escherichia coli with addition of a 6-histidine-
tag (His-tag) in order to facilitate its purification. Although
racemase as third auxiliary enzyme allowed conversion of L-Ala
3
7
into D-Ala before oxidation by readily available D-AAO.
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CP al et aa lsy es i ds oS c ni eo nt c ae d &j uTs et c mh na or gl oi ng sy
Journal Name
ARTICLE
commercially available, HPT was readily prepared from cystamine that desulfination and production of the colored thiolate were not
View Article Online
DO I- :1 10.1039/D0CY02070B
on multigram scale, using a modified procedure reported for the rate limiting. A specific activity of 0.54 U.mg was calculated for
synthesis of CSA from cystine (see SI).39 In this assay, regarding our Q9A3Q9 with this couple of substrates. The limit of detection (LOD)
2
9
previous results obtained with CSA and α-TA, we assumed that of TA activity was found at 0.02 mU/well (i.e. 0.02 nmol of substrate
HPT should be quantitatively converted into sulfite upon converted per min and per well). Moreover, the maximum
transamination whereas sulfites would be efficiently titrated with measurable activity was found at 16 mU/well. This latter value
DTNB in the presence of an amine-TA. In order to verify these corresponds to the absorbance saturation reached in the minimum
assumptions and determine the dynamic range of measurable time needed to measure a reliable activity under the experimental
enzyme activities, various concentrations of purified Q9A3Q9 (0.03 conditions used. Hence, this assay offers a dynamic range from 1 to
-
7 μg/well) were used with HPT (20 mM), Pyr (2 mM) and DTNB (1 800 which is very close to what was previously obtained with an α-
2
9
mM). Under these conditions, a linear correlation was observed TA (Asp-TA) and CSA used as sulfinic amine donor.
over the whole range of TA concentration (see figure S1), indicating
c c
, σ
, HT and activities of 20 best hits[a] in mU/well =
Table 1. Selected screening results from the direct assay with HPT as amine donor (μ
nmol SO produced/min/well).
2
CO2H
CO2H
O
Acceptor substrate
No acceptor
0
(A )
O
H
O
Pyr
PrCHO
2OP
0.002
Control mean
μ
C
0.018
0.011
0.051
0.0003
0.0002
0.001
Control standard deviation
Hit threshold
σ
HT
C
0.0005
0.003
Total hit number
64
94
24
Entry
Uniprot Id.
Annotation[b]
1
2
3
4
5
6
7
8
9
B2TF42
D0KX56
A4G7S3
E1SVL1
A0KEV7
A0P1C1
Q2JYL2
A9CF07
B1KH99
G2T7Q7
Q48AP6
E1V5R2
Q47WV7
F0SJ00
A0NZD8
C6XP14
B9Z0Z6
A3SIW8
B9K5I2
B9R1U5
B8G642
L8Q0Z4
I0JLI1
A4IMV2
B8J2Z6
A9CV07
B9L0N2
Q13M75
D4YMU7
A9DZF6
J7GK03
C0BVS9
C4G8S3
C6LGL6
A8RJD7
D9R961
Class III-TA
Class III-TA
ω-TA
Class III-TA
ω-TA
β-TA
β-TA
Am-Ao-TA
Class III-TA
β-TA
0.016
0.022
0.006
0.007
0.013
0.006
0.012
0.013
0.002
0.014
0.015
0.015
0.010
0.007
0.014
0.001
0.011
0.032
0.006
0.009
0.003
0.009
0.011
0.006
0.004
0.001
0.021
0.019
0.002
0.006
0.005
0.001
0.001
0.004
0.003
0.003
0.81
1.51
1.08
1.20
12.77
2.01
0.63
2.26
0.21
1.80
3.03
2.45
3.27
0.24
0.64
0.05
0.08
0.20
1.28
0.45
0.53
0.39
5.04
0.98
1.07
9.01
0.44
0.58
0.17
0.07
0.36
0.06
0.10
0.15
0.29
0.17
0.12
0,47
0,41
0,42
0,13
0,33
0.14
0.17
0.93
0.36
0.84
0.25
0.15
0.02
0.05
0.14
0.27
0.07
0.02
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
ω-TA
β-TA
ω-TA
β-TA
0.05
up
0.08
0.02
0.05
Class III-TA
Class III-TA
up
Am-Ao-TA
Class III-TA
Class III-TA
TA
Am-Ao-TA
Am-Ao-TA
Class III-TA
Am-Ao-TA
T-P-TA
0.15
0.13
0,50
0.58
1.04
0.45
0.03
0.01
0.16
0.02
0.05
0.01
0.09
TA
GSA
0.02
0,01
0.02
0.05
0.06
0.02
0.01
0.03
0.04
0.03
Gaba-TA
Gaba-TA
Ac-Orn-TA
Ac-Orn-TA
Ac-Orn-TA
Ac-Orn-TA
Ac-Orn-TA
[
a]
The color gradient from light to dark blue underline the relative activity of the 20 best hits obtained with each carbonyle substrate. For each selected hit, activities detected with
the other substrates are also given. [b] GSA = glutamate-1-semialdehyde aminomutase, ω-TA = ω-amino acid-pyruvate-transaminase, β-TA = β-alanine-pyruvate-transaminase, Am-
Ao-TA = adenosylmethionine-8-amino-7-oxononanoate-transaminase, up = uncharacterized protein, T-P-TA = taurine-pyruvate-transaminase, Ac-Orn-TA = acetylornithine-
transaminase, Gaba-TA = -aminobutyrate-transaminase.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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DOI: 10.1039/D0CY02070B
ARTICLE
These results confirmed that HPT can behave as a smart amine added acceptor (see A
donor for amine-TA, affording an equilibrium shift and a highly to HT and to 4*A
0
in table 1). Only TA with activities superior
were then considered as real hits.
0
efficient colorimetric assay.
Selected screening results are reported in table 1. Interestingly,
low HT (0.051 mU/well) with HPT/Pyr couple of substrates, and low
A values measured with HPT alone allowed the highly sensitive
0
Screening of an amine-TA collection with the direct assay.
In order to identify a variety of amine-TA able to use HPT as amine
donor, the direct assay was applied to the screening of a collection
of 549 well over-expressed candidate amine-TA selected from
biodiversity. The selection was done through a sequence driven
approach by genome mining, using 60 known TA from class III as
reference sequences (table S2). This collection was expected to be
representative of the diversity of amine-TA from class III (see SI for
the TA library construction). Experiments were performed on cell
free extracts in 96-well microplates and Pyr was first used as
standard acceptor. A reliable hit threshold (HT) was determined
using a statistical approach with 32 independent cultures of E. coli
host cells without overexpressed TA as negative control (see
experimental section for detailed calculation of HT). Moreover, in
order to take into account the presence of endogenous acceptors in
E. coli lysates and a possible acceptor-independent desulfination of
HPT, activity of each lysate was also measured in the absence of
detection of 64 TA able to use HPT as amine donor. The 20 best hits
outlined in table 1 displayed activities above 0.5 mU/well which
should roughly correspond to apparent specific activities above 0.1
-1
U.mg considering the mean protein quantity present in each well
(5 μg). Moreover, it appeared that hits are distributed in distinct
groups of enzymes, with various annotations and share low
homologies (see figure S4 and S5). Results obtained with two other
acceptors are also reported in table 1. Although butanal (PrCHO)
was already reported to be a substrate of several amine-TA, 2-
oxopentanoic acid (2OP) represented a more challenging substrate.
Indeed, considering the commonly described general model of (S)-
40
amine-TA active site, the propyl chain of 2OP should be poorly
accommodated in a small binding pocket where groups larger than
methyl or ethyl are rarely tolerated.
-1
[a]
Table 2. Specific activities of selected TA (in U.mg = μmol of substrate converted per min and per mg of TA).
a.
O
NH2
TA
SO2H
R1
R2
+
2
H N
R1
R2
+
+
SO2
O
Acceptor
HPT
Q48AP6
A0KEV7
I0JLI1
A9CV07
Acceptor
R1
R2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
2
2
2
2
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
H
H
1.38
1.00
0.44
0.10
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.01
0.02
0.01
0.12
0.12
0.12
0.07
0.24
0.08
1.83
2.31
0.26
0.17
0.02
0.04
0.02
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.06
0.04
0.03
0.02
0.14
0.89
0.76
0.45
0.22
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.01
0.02
0.02
0.25
0.15
0.14
0.07
0.35
0.06
1.69
3.00
0.30
0.65
Me
CH
Et
Pr
Bu
CH
iPr
iBu
CH
Me
Ph
CH
CH
H
Me
Et
2
OH
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.04
2
CH
2
SMe
0
1
2
3
4
5
6
7
8
9
0
1
2
CH
2 2
CO H
Me
Me
Me
2
OH
OH
CH
H
2
OH
2
H
H
H
H
H
H
Pr
Ph
CH
CH(OH)CH
2
OH
2
OH
0.02
0.01
[
a]
Reaction conditions: acceptor (2 mM), HPT (10 mM), PLP (0.1 mM), DTNB (1 mM), TA (1-80 μg/mL), DMSO (5%), potassium phosphate buffer (50 mM, pH 7.5)
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DOI: 10.1039/D0CY02070B
ARTICLE
Low HT values, determined for both substrates allowed the ID A0A0H3K2P4 previously referenced Q5N2G0) as a model TA. This
2
9
detection of 94 and 24 hits for PrCHO and 2OP respectively, which enzyme was identified in previous screening experiments, and
were all also active with Pyr as acceptor. With PrCHO, the 20 best was found to display a good activity with Ala and 2OG while being
hits displayed activities from 0.12 to 1.04 mU/well. In the case of completely inactive with HPT as amine donor. A calibration curve
2
OP, as anticipated, low activities were measured with only two hits was established in screening conditions, using various
over 0.1 mU/well. These two TA were also found among the 20 best concentrations of model TA A0A0H3K2P4 (0.6 - 47 μg/well), Ala (20
hits detected with Pyr and PrCHO (table 1, entries 4 and 5). These mM), HPT (10 mM), KG (2 mM), DTNB (1 mM) and auxiliary TA
results showed that the direct assay is suitable for the highly A9CV07 (6.7 μg, 20 mU/well). Under these conditions, as shown in
sensitive detection in cell lysates of amine–TA able to use HPT as figure 2, linearity was observed at least up to 1 mU/well and an
amine donor and a variety of acceptor substrates.
activity LOD of 0.05 mU/well was calculated. A dynamic range from
to 20 was thus obtained in these conditions which were deemed
suitable for large scale screening experiments.
1
Coupled assay performance and validation.
Because HPT is clearly not a standard amine donor for TA, and
considering that Ala is a substrate of most amine–TA described to
Screening of an amine-TA collection with the coupled assay.
date, we designed the coupled assay in which Ala is used as primary Representative results of the screening of our TA collection with the
newly developed coupled assay are reported in table 3. As
discussed above about the screening using the direct assay, hit
thresholds were accurately determined through a series of control
experiments without overexpressed TA or acceptor substrate. In
order to highlight the complementarity of both assays, we decided
to use the same carbonyl derivatives, PrCHO and 2OP as acceptor
substrates in the presence of Ala as primary amine donor.
amine donor and is regenerated using an auxiliary TA and HPT as
secondary amine donor. First experiments with Q9A3Q9 used for
direct assay development rapidly showed that this TA was neither
active nor stable enough for use in large scale screening
experiments. Therefore, an alternative auxiliary HPT-TA was
searched among the hits from the first screening of our TA
collection performed with the direct assay. Hence, four TA among
the most active with Pyr and HPT, A0KEV7, Q48AP6, I0JLI1 and
A9CV07 (table 1, entries 5, 11, 23 and 26) were recloned with
Most of the TA already found as hits using the direct assay
were logically detected again with the coupled assay. Nevertheless,
1
0
8 TA displaying low activities with 2OP and HPT as unique donor (<
.1 mU/well), were not detected with the coupled assay because of
addition of
a His-tag, produced and purified for further
characterisation. Specific activities of these TA were measured,
a higher HT (0.132 mU/well). This relatively high value likely results
using the direct assay, for a series of 21 acceptors and are reported from the activity of host cells constitutive α-TA able to use Ala and
in table 2. All of the four TA showed activities higher than 0.1 U/mg 2OP as substrate and justify the limited number of hits (15)
with short chain keto acids 1-4 with A9CV07 being the most active detected with 2OP using the coupled assay.
enzyme with Pyr 2. A low activity (< 0.05 U/mg) with keto acids 5-7
bearing side chains with more than 2 carbons was detected with
A0KEV7 only. None of the TA showed detectable activity with
branched or carboxylated keto acids 8-10, acetone 11 or
acetophenone 12. A low activity (< 0.02 U/mg) was observed with
hydroxyacetone, dihydroxyacetone and formaldehyde 13-15 in the
case of Q48AP6 and I0JLI1 whereas all other tested aldehydes 16-21
were shown to be substrates of Q48AP6, A0KEV7 and I0JLI1. Finally,
A9CV07 was shown to display a low activity only with hydroxylated
aldehydes 20 and 21. Therefore due to its good activity with Pyr and
its narrow substrate spectrum, A9CV07 from Hoeflea
phototrophica, appeared as the best candidate for use as auxiliary
enzyme in the coupled assay. Satisfactorily, thermostability studies
showed that this TA maintained 80 % of its activity after 48 h at 30
°
C (see Fig S2). Subsequently, the direct assay was used to
accurately determine apparent Michaelis constants (Km) and kcat for
the transamination reaction between HPT and Pyr catalysed by
A9CV07 (see Fig S3): KmHPT = 22.0 ± 1.2 mM, KmPyr = 0.25 ± 0.03
-1
mM, kcat = 181 ± 7 min . Having identified a suitable auxiliary TA,
transamination between 2-oxoglutaric acid (2OG) and Ala was
chosen as model reaction and a TA from Synechococcus sp. (Uniprot
Figure 2. Calibration curve of the model TA with the coupled assay
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CP al et aa lsy es i ds oS c ni eo nt c ae d &j uTs et c mh na or gl oi ng sy
Page 6 of 9
View Article Online
DOI: 10.1039/D0CY02070B
ARTICLE
Table 3. Selected screening results from the coupled assay with L-Ala as primary amine donor (μ
in nmol SO produced/min/well).
c
, σ
c
, HT and activities of the 20 best hits[a]
2
CO2H
No acceptor
Acceptor substrate
O
H
O
0
(A )
PrCHO
2OP
Control mean
Control standard deviation
Hit threshold
μ
C
0.016
0.004
0.029
0.076
0.007
0.132
σ
C
HT
Total hit number
114
15
Entry
Uniprot Id.
A7HSN3
A3SLG7
E1SVK1
A0KQL1
C6XP14
B9Z0Z6
B9JUL0
A4ABK4
A9EB31
A3SIW8
Q1MXW4
E1V9D2
F0SJ00
D4XGV2
Q48AP6
Q47WV7
E1SVL1
A0KEV7
A0P0G6
A3K8H0
L8Q0Z4
I0JLI1
Annotation[b]
Class III-TA
up
Class III-TA
Class III-TA
Class III-TA
Class III-TA
Am-Ao-TA
Am-Ao-TA
TA
up
up
up
β-TA
Am-Ao-TA
ω-TA
ω-TA
Class III-TA
ω-TA
up
up
TA
Am-Ao-TA
T-P-TA
Gaba-TA
Pu-TA
Ao-TA
Ao-TA
Ao-TA
1
2
3
4
5
6
7
8
9
0.011
0.057
0.046
0.079
0.008
0.024
0.006
0.047
0.013
0.036
0.012
0.033
0.013
0.018
0.014
0.010
0.015
0.019
0.060
0.035
0.003
0.005
0.023
0.028
0.009
0.009
0.010
0.011
0.85
1.11
1.61
5.26
1.24
2.54
0.62
0.72
1.17
1.88
1.75
0.42
0.70
0.71
1.04
0.90
0.35
0.30
2.43
0.66
0.96
1.14
0.86
0.19
0.51
1.01
1.24
0.29
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
0.19
0.18
0.36
0.16
B9L0N2
B2JR37
B5Y8A4
A1HTU7
C0BVS9
C6LGL6
0.17
0.24
0.16
0.83
0.18
0.15
0.04
0.49
[
a]
The color gradient from light to dark blue underline the relative activity of the 20 best hits obtained with each substrate. For each selected hit, activities detected with other
substrates are also given. [b] ω-TA = ω-amino acid-pyruvate-transaminase, β-TA = β-alanine-pyruvate-transaminase, Am-Ao-TA = adenosylmethionine-8-amino-7-oxononanoate-
transaminase, up = uncharacterized protein, T-P-TA = taurine-pyruvate-transaminase, Ao-TA = acetylornithine-transaminase, Pu-TA = putrescine-aminotransferase, GABA-TA = -
aminobutyrate-transaminase.
Although close activities were often measured with both assays, therefore offering the best opportunities for synthetic applications.
(see for example entries 15, 16 and 22 with PrCHO and entries 17 Finally, as outlined for the direct assay, the newly identified amine-
and 18 with 2OP), higher values were measured with the coupled TA showed broad diversity (see figure S4 and S5) and constitute an
assay in several cases (see for example entries 5,10 and 23 with unprecedented set of biocatalysts with high synthetic potential for
PrCHO and entry 6 with 2OP) indicating that Ala is a better donor the highly stereoselective synthesis of chiral amines.
than HPT for these particular TA. More interestingly, and as
expected, many hits were exclusively detected with the coupled
assay and correspond to enzymes inactive with HPT but accepting
Experimental Section
Ala as amine donor. Hence, 10 out of the 20 best hits with PrCHO Screening of the TA library. All reactions assays were performed at
and 9 out of the 15 hits with 2OP were detected only with the 30 °C, in 50 mM potassium phosphate buffer, pH 7.5, in 96-well
coupled assay. Moreover, for both acceptors, the coupled assay microplates and in a total volume of 200 µL. The direct assay was
allowed the detection of TA displaying higher activities and implemented using 10 mM HPT, 2 mM carbonyle acceptor, 0.05
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CP al et aa lsy es i ds oS c ni eo nt c ae d &j uTs et c mh na or gl oi ng sy
Journal Name
ARTICLE
4
F. Steffen-Munsberg, C. Vickers, H. Kohls, H. LaVniedw, AHrt.icMle Oalnlliinne,
mM PLP, 1 mM DTNB, 1% EtOH, and 5 μL of cell lysate from the TA
library. The coupled assay was implemented using 20 mM L-Ala, 10
mM HPT, 2 mM acceptor, 0.05 mM PLP, 1 mM DTNB, 1 % EtOH, 20
mU/well A9CV07 (6,7 μg), and 5 μL of cell lysate from the TA library.
Lysates from 32 independent cultures of E.coli cells bearing an
empty plasmid were used in the same conditions as negative
control for all type of assays. These control experiments allowed to
A. Nobili, L. Skalden, T. van den Bergh, H.-J. Joosten, P.
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Neto and J. M. Woodley, Biotechnol. Bioeng., 2011, 108,
5
6
7
calculate means (μ
C
), standard deviations (σ
C
) and hit threshold (HT
) for each set of
1
479.
=
μ
C
+ 3*σ or HT = 1,75*μ
C
9
C
if σ < 0.25*μ
C
C
8
9
R. J. Meier, M. T. Gundersen, J. M. Woodley and M.
Schürmann, ChemCatChem, 2015, 7, 2594.
2
substrates. Another control experiment was performed for both
direct and coupled assays and for each enzyme of the library in the
C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam,
W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, et
al., Science, 2010, 329, 305.
same conditions but without any acceptor substrate (A
were stirred 90 s before absorbance variations were recorded at
12 nm over 30 min. TA activities were calculated using ε = 14150
0
). Plates
1
0 E.-S. Park, J.-Y. Dong and J.-S. Shin, Org. Biomol. Chem., 2013,
4
1
1, 6929.
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-1
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Wang and W. Wu, CHIMIA, 1999, 53, 584.
0
above both HT and 4*A were considered as hits.
1
1
2 F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler and W.
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3 M. D. Truppo and N. J. Turner, Org. Biomol. Chem., 2010, 8,
Conclusions
1
280.
We have developed two new continuous colorimetric assays 14 S. Schätzle, F. Steffen-Munsberg, A. Thontowi, M. Höhne, K.
suitable for screening amine-TA collections. Both assays,
implemented on crude cell lysates, displayed high sensitivity and
broad dynamic range. The direct assay allowed the identification of
a series of highly active HPT-TA, among which an enzyme with a
narrow substrate spectrum proved suitable for use as the auxiliary
Robins and U. T. Bornscheuer, Adv. Synth. Catal., 2011, 353,
439.
5 H.-H. Lo, S.-K. Hsu, W.-D. Lin, N.-L. Chan and W.-H. Hsu,
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6 T. Li, A. B. Kootstra and I. G. Fotheringham, Org. Process Res.
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2
1
1
enzyme in the coupled assay. The latter was proven useful for the 17 A. P. Green, N. J. Turner and E. O’Reilly, Angew. Chem. Int.
Ed., 2014, 53, 10714.
reliable TA activity measurement with L-Ala as standard amine
1
1
8 L. Martínez‐Montero, V. Gotor, V. Gotor‐Fernández and I.
Lavandera, Adv. Synth. Catal., 2016, 358, 1618.
9 A. Gomm, W. Lewis, A. P. Green and E. O’Reilly, Chem. Eur. J.,
2016, 22, 12692.
donor and various carbonyl acceptors. Moreover, the coupled assay
can obviously be implemented in a stereoselective fashion by using
L- or D-Ala as amine donor in order to distinguish (S)- or (R)-amine-
TA. Finally the transamination reaction in both assays is the 20 I. Slabu, J. L. Galman, N. J. Weise, R. C. Lloyd and N. J. Turner,
ChemCatChem, 2016, 8, 1038.
conversion of interest in asymmetric synthesis, i.e. the
2
2
2
1 R. C. Simon, F. Zepeck and W. Kroutil, Chem. Eur. J., 2013, 19,
enantioselective formation of a chiral amine from a prochiral
ketone, thus giving a reliable prediction of the TA applicability in
biocatalytic processes.
2
859.
2 E. Heuson, F. Charmantray, J.-L. Petit, V. de Berardinis and T.
Gefflaut, Adv. Synth. Catal., 2019, 361, 778.
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2
Conflicts of interest
There are no conflicts to declare.
2
Acknowledgements
We thank the French National Center for Scientific Research
2
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2
8 G. L. Ellman, Arch. Biochem. Biophys., 1959, 82, 70.
9 E. Heuson, J.-L. Petit, A. Debard, A. Job, F. Charmantray, V.
de Berardinis and T. Gefflaut, Appl. Microbiol. Biotechnol.,
(CNRS) and University of Clermont-Auvergne for financial
support. We also thank Ms Martine Sancelme for enzyme
production and Ms Virginie Pellouin for technical assistance.
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ARTICLE
Journal Name
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| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Catalysis Science & Technology
Entry for the Table of Contents
View Article Online
DOI: 10.1039/D0CY02070B
Versatile and sensitive continuous colorimetric assays were developed for the high throughput screening of a large collection of
amine-TA from biodiversity, and allowed the discovery of a new set of diverse biocatalysts with high synthetic potential.