A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases

Article abstract of DOI:10.1039/c0cc02919j

A fast and sensitive method for screening transaminase activity and enantioselectivity, using d- and l-amino acid oxidases, allows new amine substrates to be rapidly identified. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0cc02919j

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A fast and sensitive assay for measuring the activity and
enantioselectivity of transaminaseswz
Jennifer Hopwood, Matthew D. Truppo, Nicholas J. Turner* and Richard C. Lloyd
a
a
a
b
Received 30th July 2010, Accepted 19th October 2010
DOI: 10.1039/c0cc02919j
A fast and sensitive method for screening transaminase activity
and enantioselectivity, using D- and L-amino acid oxidases,
allows new amine substrates to be rapidly identified.
to L- or D-alanine 3 which is detected by the addition of an
amino acid oxidase (L- or D-AAO respectively) to the reaction
mixture. Oxidation of alanine 3 to the imine 4 by AAO results
2 2
in the production of H O which can be detected colori-
Amino transaminases (ATAs) are pyridoxal phosphate
dependent enzymes that catalyse the reversible transfer of
ammonia from an amine donor to a ketone acceptor. Recently
they have attracted considerable interest as biocatalysts for the
metrically by the addition of pyrogallol red and horse radish
6
peroxidase. Provided that all subsequent enzymes and reagents
are added in excess then the ATA step becomes rate-limiting
1
Table 1 Relative activity of amines with transaminase ATA-117
using the D-AAO based colorimetric assay
synthesis of enantiomerically pure chiral primary amines.
Particular attention has been paid to (i) overcoming problems
of substrate/product inhibition, (ii) solving issues related to
poor equilibrium conversions of ketone to amine and (iii)
Relative
initial rate/% Amine
Relative
initial rate/%
Amine
2
developing ATAs with broader substrate specificity. Equally
important is the ability to be able to rapidly screen ATAs for
new substrates, with respect to both activity and enantio-
selectivity. Previous methods for screening for ATA activity
have been based upon monitoring pH changes using the
1
39
51
23
2
3
indicator dye phenyl red, formation of coloured Cu–alanine
4
complexes and also an elegant UV spectrophotometric
5
protocol. Herein we describe a simple colorimetric-based method
123
and show that it can be used to determine both the activity and
enantioselectivity of a wide range of ATA substrates.
Scheme 1 outlines the basis for the new screening method.
Amines 1 are treated with an ATA and sodium pyruvate 2.
Successful substrates result in conversion of sodium pyruvate
a
1
00
9
6
1
9
7
2
3
1
0
Scheme 1 (i) Transaminase (ATA); (ii) D- or L-amino acid oxidase
(AAO); (iii) horse radish peroxidase (HRP); (iv) pyrogallol red (PGR).
a
School of Chemistry, University of Manchester,
Manchester Interdisciplinary Biocentre, 131 Princess Street,
Manchester M17DN, UK.
E-mail: Nicholas.turner@manchester.ac.uk;
Fax: +44 (0)161 306 5173
Dr Reddy’s, Chirotech Technology Ltd, Unit 162,
Cambridge Science Park, Milton Road, Cambridge CB4 0GH, UK.
E-mail: rlloyd@drreddys.com; Fax: +44 (0)1223 506701;
Tel: +44 (0)1223 728010
6
6
6
6
0
0
b
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
z Electronic supplementary information (ESI) available: Assay
conditions: sample preparation for GC and examples of spectra. See
DOI: 10.1039/c0cc02919j
a
%
initial rates are relative to a-methylbenzylamine.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 773–775 773

Fig. 1 Comparison of relative initial rates (relative to a-methyl-
benzylamine 7) of transamination, with five different substrates, in the
forward (ketone to amine: GC) and reverse (plate reader) directions.
Scheme 2 Determination of the enantioselectivity of the trans-
aminase catalysed reactions by using (a) D- or (b) L-AAO with the
and hence its activity with respect to the amine substrate of
interest can be determined.
(
R)- or (S)-7 amine substrate respectively.
To assess the generality of this screening method we
examined a panel of 16 different amines with the (R)-selective
7
,8
ATA-117 and D-AAO (Table 1). The entire set of substrates
was screened at 5 mM concentration to obtain relative initial
rates. Using this approach we were able to confidently determine
initial rates as low as 1% compared to that of amine 7.
In order to establish that the rates of the ATA catalysed
reactions determined by this assay in the cleavage direction
(
amine to ketone) were comparable to the rates measured in
the synthesis direction (ketone to amine), we selected five
amine substrates 6, 7, 8, 12 and 13 for study. For each
substrate the initial rate (relative toa-methylbenzylamine 7)
of synthesis of the amine from the corresponding ketone was
9
measured using a conventional GC based assay and compared
Fig. 2 Evaluation of the stereoselectivity of transamination reactions
with either (R)- or (S)-7 as substrate. For experiment with (R)-7,
D-AAO was used in the assay whereas with (S)-7, L-AAO was used in
the assay.
to the initial rates determined by the colorimetric assay. Fig. 1
shows that generally good correlation between the forward
(
GC) and reverse (colorimetric assay) rates of transamination
was obtained confirming that the current method provides an
excellent way of indirectly assessing activity in the synthesis
direction. The disparity between the rates for compound
results in a very low initial rate, confirming the high stereo-
selectivity of ATA-117 towards (R)-amines. Control experiments
showed that both L-AAO and D-AAO were highly enantio-
selective for L-alanine and D-alanine respectively. Therefore,
the enantioselectivity of both ATA-113 and ATA-117 (in this
example 87% ee and 99% ee respectively) can be simply
estimated by comparing the initial rates of each enzyme for
transamination of (R)- versus (S)-7.
6
is currently unclear but may be due to differences in the
solubility of the ketone and amine substrates.
In addition to information about relative rates of transamina-
tion it is also desirable to determine the levels of enantio-
selectivity of the reaction. The colorimetric assay can be easily
adapted in the following way to collect such data rapidly
(
Scheme 2).
In summary, we have developed a facile and rapid method
for screening for transaminase activity and enantioselectivity
which uses inexpensive and readily available reagents and
moreover only requires a UV/Vis-plate reader to operate in
96-well microtitre plate format. The further development of
this assay for high-throughput screening of transaminase
libraries is currently under investigation.
In the first experiment (Scheme 2a) we used (R)-7 as
substrate with D-AAO as the reagent to detect production of
D-alanine as described above. Using transaminase ATA-117,
which is known to be selective for (R)-configured amines, the
reaction generates D-alanine and hence a signal with high
intensity is observed due to the high rate of reaction (Fig. 2).
If ATA-117 is replaced by the (S)-selective ATA-113 then the
signal intensity is considerably reduced although there is
indeed a measurable level of conversion. This low level of
activity suggests that ATA-113 is able to catalyse transamina-
tion of (R)-7, although to a reduced amount, and corres-
pondingly generate D-alanine. By comparison, when (S)-7 is
used as the substrate (Scheme 2b), with L-AAO as the enzyme
for detection of L-alanine, a high initial rate is detected with
ATA-113 as expected. Replacing ATA-113 with ATA-117
Notes and references
1
N. J. Turner and M. D. Truppo, Biocatalytic routes to non-racemic
chiral amines, in Chiral Amine Synthesis, ed. T. C. Nugent, Wiley,
Weinheim, 2010, pp. 431–457; M. Hoehne and U. T. Bornscheuer,
ChemCatChem, 2009, 1, 42.
2
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,
P. N. Devine, G. W. Huisman and G. J. Hughes, Science, 2010, 329,
305; M. D. Truppo, J. D. Rozzell and N. J. Turner, Org. Process
7
74 Chem. Commun., 2011, 47, 773–775
This journal is c The Royal Society of Chemistry 2011

Res. Dev., 2010, 14, 234; M. D. Truppo, J. D. Rozzell and
N. J. Turner, Chem. Commun., 2009, 2127; D. Koszelewski,
I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell and
W. Kroutil, Angew. Chem., Int. Ed., 2008, 47, 9337;
D. Koszelewski, I. D. Clay, K. Faber and W. Kroutil, J. Mol.
Catal. B: Enzym., 2009, 60, 191; D. Koszelewski, I. Lavandera,
D. Clay, D. Rozzell and W. Kroutil, Adv. Synth. Catal., 2008, 350,
D-amino acid oxidase from porcine kidney (D-AAO-A5222) were
purchased from Sigma-Aldrich.
8 Assay conditions: a stock solution of substrate and reagents was
ꢀ
1
ꢀ1
made up as follows; 0.1 g L of PGR, 0.2 g L of sodium
ꢀ
1
pyruvate, and 0.05 g L of PLP were added to 1 L of 100 mM
phosphate buffer. 50 mL of this stock solution and 5 mM of amine
donor (D- or L-alanine) were added to a falcon tube and the
pH adjusted to 8. To 100 mL of this solution was added 10 mL of
2761; H. Yun and B.-G. Kim, Biosci., Biotechnol., Biochem., 2008,
ꢀ1
72, 3030; D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell and
HRP (1 g L ), 20 mL of D-AAO (60 U). Finally, 100 mL of the
resulting solution was dispensed into individual wells in a 96-well
MTP and assays were initiated by the addition of 2.5 mL of
W. Kroutil, Adv. Synth. Catal., 2008, 350, 2761; H. Yun and
B.-G. Kim, Biosci., Biotechnol., Biochem., 2008, 72, 3030;
ꢀ1
ꢀ1
M. Ho
¨
008, 350, 807; M. Ho
hne, K. Robins and U. T. Bornscheuer, Adv. Synth. Catal.,
hne, S. Kuhl, K. Robins and
ATA-117 (0.6 g L ) or ATA-113 (0.6 g L ). Experiments were
run on a spectrophotometer at 30 1C with a wavelength of 540 nm
and absorbance readings taken every 30 seconds.
2
¨
¨
U. T. Bornscheuer, ChemBioChem, 2008, 9, 363; R. L. Hanson,
B. L. Davis, Y. Chen, S. L. Goldberg, W. L. Parker, T. P. Tully,
M. A. Montana and R. N. Patel, Adv. Synth. Catal., 2008,
9 GC assay conditions: an LDH/GDH system was employed to run a
transamination in the forward direction. 10 g L of glucose, 45 g
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
350, 1367.
L of D-alanine, 1 g L of NAD and 0.5 g L of PLP were added
to 16 mL of 100 mM phosphate buffer and the pH adjusted to
7.5 using sodium hydroxide (NaOH). 1 g L of LDH, 1 g L of
3
4
5
6
M. D. Truppo, D. J. Rozzell, J. C. Moore and N. J. Turner, Org.
Biomol. Chem., 2009, 7, 395.
B.-Y. Huang and B.-G. Kim, Enzyme Microb. Technol., 2004, 34,
ꢀ
1
ꢀ1
ꢀ
1
GDH and 5 g L of ATA-117 was then added to the stock solution.
0.8 mL of the stock solution was added to five different Eppendorf
tubes along with 200 mL of DMSO and separately 5 mM of
acetophenone, 4-chloro-acetophenone, 4-bromoacteophenone,
4-acteylpyridine and 3-acteylpyridine were added. The solutions
were heated to 30 1C and agitated at 900 rpmand samples taken
after 5 hours. 40 mL of 5 M NaOH was added to stop the reaction
and the pH adjusted to 7 followed by extraction (3ꢁ) with 1 mL of
chloroform. The remaining solution was concentrated to 1 mL and
429.
S. Scha
¨
¨
tzle, M. Hohne, E. Redestad, K. Robins and
U. T. Bornscheuer, Anal. Chem., 2009, 81, 8244.
H. Iloukhani, F. Rajali and A. Zeini, Phys. Chem. Liq., 2001, 39,
2
1
39; A. A. Ensafia and B. Rezaeia, Anal. Lett., 1993, 26,
771.
7
Enzymes including transaminases (ATA-113 and ATA-117), GDH
and LDH were generously supplied by Codexis (Redwood City, CA).
L-Amino acid oxidase from Crotalus adamanteus (L-AAO-A9378) and
3 2
the amine acetylated with Et N : Ac O (2 : 1) and analysed via GC.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 773–775 775