A high-throughput screening method for determining the substrate scope of nitrilases

Article abstract of DOI:10.1039/c4cc06021k

Nitrile compounds are intermediates in the synthesis of pharmaceuticals such as atorvastatin. We have developed a chromogenic reagent to screen for nitrilase activity as an alternative to Nessler's reagent. It produces a semi-quantifiable blue colour and hydrolysis of 38 nitrile substrates by 23 nitrilases as cell-free extracts has been shown. This journal is

Full text of DOI:10.1039/c4cc06021k

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A high-throughput screening method for determining
the substrate scope of nitrilases.
Cite this: DOI: 10.1039/x0xx00000x
a
a
a
b
Gary W. Black , Nicola L. Brown , Justin J. B. Perry *, P. David Randall , Graeme
a
a
Turnbull and Meng Zhang
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
7
Nitrile compounds are intermediates in the synthesis of chromophore to measure acid generation and the formation of a
1
8
chromophore or fluorophore to measure ammonia generation,
pharmaceuticals such as atorvastatin. We have developed a
chromogenic reagent to screen for nitrilase activity as an
however in each case activity is measured using whole live cells or
purified enzymes. The use of lyophilised cleared cell lysates (usually
referred to as cellꢀfree extracts, CFEs) provides a convenient way of
screening potential new nitrilases for substrate specificity without
alternative to Nessler’s reagent. It produces
a semi-
quantifiable blue colour and hydrolysis of 38 nitrile
substrates by 23 nitrilases as cell-free extracts has been the need for enzyme purification, however current rapid screening
shown.
methods are incompatible with cellular components present in CFE
solutions. Here we present a facile and inexpensive highꢀthroughput
Nitrile compounds (RꢀC≡N) are useful intermediates in the synthesis method for the screening of nitrilaseꢀcontaining CFE libraries
1
of fine chemicals and pharmaceuticals such as atorvastatin, however against a wide range of nitrile substrates, which utilises the
chemical hydrolysis of these compounds usually requires harsh chromogenic reaction between oꢀphthalaldehyde (OPA, 1) and
2
conditions, such as extremes of pH and high temperatures with the ammonia under acidꢀcatalysed conditions (Scheme 1), and report its
3
creation of significant quantities of byproducts. Biocatalytic use to indicate the substrate preferences of a library of nitrilases
hydrolysis is seen as a viable ‘green’ alternative for the production against a range of structurallyꢀdiverse substrates.
of these acids as it can be conducted at nearꢀambient temperature
3,4
and neutral pH. Nitrileꢀhydrolysing enzymes typically display
enantioꢀ and regioselectivity that simply cannot be achieved through
3
,5–7
chemical hydrolysis.
There are two classes of nitrileꢀhydrolysing
enzymes: nitrile hydratases (EC 4.2.1.84) which convert nitrile
8
functionality to primary amides; and nitrilases (EC 3.5.5.1) which
9
convert nitriles to the related carboxylic acid.
The pace of development of novel biocatalytic processes has
increased with advances in genomics, laboratoryꢀbased evolution
1
0–12
and protein expression,
however this is limited by the rate at
which new enzymes can be screened for activity and substrate
specificity. Methods of screening nitrilase activity in whole cells
typically involve cell culture in the presence of a range of nitrile
substrates and measurement of products by chromatographic
Scheme 1. Nitrilase hydrolyses nitrile compounds to the
corresponding carboxylic acid and ammonia. The ammonia reacts
with 1 in our reagent to generate a blue chromophore.
1
3
methods such as LCꢀ or GCꢀMS. Due to the timeꢀconsuming
nature of such work, substrate ranges are often limited to a handful
of nitrile types. Other spectroscopic techniques such as NMR and IR
spectroscopy have also found use for kinetic assays, but not for highꢀ
Banerjee et al. have reported a method that utilises 1 in the presence
19
of mercaptoethanol to generate a fluorescent product. We found
this method to be incompatible with CFE as it generated high
background fluorescence. The formation of chromogenic products in
the reaction between ammonia and 1 in the absence of thiol reducing
1
4
throughput applications.
While published reviews compare
substrate specificity for some organisms, these results are obtained
through different experimental conditions making close comparison
20
agents is well known, and accounts exist as far back as the early
4,10,15
difficult.
Several highꢀthroughput assays for nitrilase activity have been
21–23
twentieth century.
The products of this reaction are known to be
24
unstable and are thought to be the result of several competing
reactions occurring in parallel,
1
6
developed, including the use of pH indicators or formation of a
22,25,26
though an isoindole species is
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DOI: 10.1039/C C0602
Jou4Crnal Na1Kme
believed to be the most abundant product. Despite this lack of clarity
regarding the nature of the chromophoric species, it can be shown to
be suitable to form the basis of an ammoniaꢀdetecting assay suitable
aliphaticsubstrates
for
semiꢀquantitatively
measuring
nitrilase
activity.
N
N
N
N
Reaction of 1 with ammonia in basic buffer forms a colourless
intermediate species, which on acidification becomes intensely
coloured. The hue depends on the acid used and ranges from light
blue to deep purple in colour (Fig. S1), however trichloroacetic acid
1
2
3
4
N
N
N
N
(TCA) was found to give the greatest contrast as a blueꢀblack
5
6
7
8
chromophore is formed. This chromophore can precipitate on
standing, however dilution with DMSO yields a blue species that is
suitable for quantitative measurement by UV/visible spectroscopy.
N
Using a set of standard NH Cl solutions it was shown that
4
9
quantitation of the ammonia over the range 1ꢀ11 mM was possible
2
(
R =0.999) enabling nitrilase assays to be designed which yield
aromaticsubstrates
ammonia in this concentration range (Fig. 1).
OH
HO
N
N
N
N
1
0
11
12
13
O
Cl
N
N
N
N
N
Figure 1. Reaction of OPA reagent with ammonia (concentration of
14
15
16
17
NH Cl standard solution in mM indicated as number) after dilution
4
with DMSO.
N
Cl
NH
2
F
3
C
H
To demonstrate the utility of this detection method for
determination of nitrilase activity, this screening method was
used to determine the substrate specificity and relative activity
for a range of twentyꢀthree nitrilases recombinantly expressed
in Escherichia coli (18 were rationally sampled from the
diversity that exists within sequence space [Fig. S3], and five
were commercially available through Prozomix Ltd., UK) and
stored as lyophilised CFEs (all entries are provided in the
supplementary information, Tables S1 and S2).
Solutions of these CFEs were prepared in phosphate buffer (10
mM, pH 7.2), combined with a structurallyꢀdiverse panel of
thirtyꢀeight aliphatic, aromatic and arylaliphatic nitrile
compounds (fig. 2, prepared in EtOH or DMSO to aid
solubility) in 96ꢀwell microplates and incubated overnight (37
N
N
N
N
Cl
1
8
19
20
2
1
2
N
N
N
N
N
N
N
N
2
2
23
24
25
arylaliphaticsubstrates
N
N
N
ºC, 18 h). OPA reagent was prepared as a solution of
methanol and diluted 1:100 into sodium tetraborate buffer (15
mM, pH 9.5). This reagent was combined with the incubated
1 in
O N
Cl
28
2
2
6
27
Cl
O
mixture, added to DMSO and acidified with TCA solution (aq.
N
w
1
0% /v) to yield a blueꢀblack chromogen in proportion to the
N
N
ammonia liberated by the nitrilaseꢀcatalysed hydrolysis in each
well. These solutions were further diluted with DMSO to
enable concentration determination by UV/visible spectroscopy
using a standard curve for calibration. An example of a
NO2
Cl
30
29
31
OH
microplate containing an assay of this type is shown in Fig. 3
Nine of the 23 enzymes hydrolyse one or more of these
substrates at the level of ≥20% conversion. Of these active
.
N
N
N
3
2
33
34
enzymes, five (entries 2-4
arylacetonitrilases and four (entries
,
6-7
)
1
are predominantly
, 5, 19 and 22) are
N
predominantly aromatic nitrilases. Promiscuity is however
observed across the range, with entries and 19 most active for
the widest range of substrates. Six highly active nitrilases are
immediately obvious from these results, with the
N
4
N
3
5
36
37
Bradyrhizobium spp.
3 and 4 displaying up to 100% conversion
of 2ꢀphenylacetonitrile.
Figure 2. Nitrile substrates used to screen each nitrilase CFE.
2
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DOI: 10.1039/C4CC060 K
variety of nitrile substrates in a simple, highꢀthroughput method
compatible with using a microplate reader. The ability to screen
in such small volumes provides a fast and economical way of
experimentally identifying new nitrilase enzymes, and rapidly
ascertain their substrate specificity. In comparison to the
existing Nessler assay, this reagent is free of mercury salts and
is not persistent in the environment.
Notes and references
a
Industrial Biotechnology Research Group, Department of Applied
Sciences, Faculty of Health and Life Sciences, Northumbria University,
Newcastle upon Tyne, NE1 8ST, U.K.
b
Chemoxy International Ltd, Middlesbrough, Cleveland, TS3 6AF, UK.
†
Confirmatory tests indicating the presence of carboxylic acid after
nitrile hydrolysis were performed using HPLC and GCꢀMS.
† Experimental data is included in the ESI.
†
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/c000000x/
Figure 3. Reaction of OPA reagent with nitrilase
solution after incubation with nitrile substrates 31 in triplicate
see fig. S2 for full details).
Nitrilases from Chaetomium globosum
) and PROꢀNITR010 We wish to extend our thanks to Dr. Simon Charnock of Prozomix Ltd.
19) have an obvious preference for 4ꢀsubstituted aromatic for providing the commercially available nitrilases used in this screen, to
5 CFE
1
ꢀ
(
(
5
(
nitrile compounds, while these and PROꢀNITR014 (22) display
high activity towards pyridinecarbonitriles.†
Nitrilases from species Bradyrhizobium sp. BTAi1 (A5ETE9;
Biocatalysts Ltd. for their collaboration and to the Technology Strategy
Board for funding.
8
), Rhodopseudomonas palustris (Q2J474; 9), Silicibactor
1
.
U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C.
promeroyi (Q5LLB2; 10), PROꢀNITR012 (21), and PROꢀ
NITR018 (23) displayed very weak activity against the
Moore, and K. Robins, Nature, 2012, 485, 185–94.
substrate panel (<20% substrate hydrolysed). Nitrilases from 2. M. Kobayashi and S. Shimizu, Curr. Opin. Chem. Biol., 2000,
4, 95–
species Achromobacter xylosoxidans (E3HN55), Acidovorax
avenae (F0Q9Y1), Alcaligenes faecalis (G8CXY5), Bacillus
cereus (Q819F0), Clostridium kluyveri (A5MYU1), Lachancea
thermotolerans (C5DH06), Sphaerobacter thermophilus
1
02.
3
4
.
.
A. Banerjee, R. Sharma, and U. C. Banerjee, Appl. Microbiol.
Biotechnol., 2002, 60, 33–44.
R. N. Thuku, D. Brady, M. J. Benedik, and B. T. Sewell, J. Appl.
Microbiol., 2009, 106, 703–727.
(
1
D1C8L7) and Vanderwaltozyma polyspora (A7TP07) (entries
1-18 respectively) displayed no detectable activity against the
substrate panel (<10% substrate hydrolysed).
Three CFEs (entries and 10) were screened in duplicate
5. F. B. Cooling, S. K. Fager, R. D. Fallon, P. W. Folsom, F. G.
Gallagher, J. E. Gavagan, E. C. Hann, F. E. Herkes, R. L. Phillips, A.
E. Sigmund, L. W. Wagner, W. Wu, and R. DiCosimo, J. Mol. Catal.
B Enzym., 2001, 11, 295–306.
3, 6
from separately prepared cultures. While determination of
substrate preference was repeatable the absolute conversion
was not identical, highlighting a disadvantage of using CFEs
over purified enzymes where activity per gramme is a tightly
defined value in comparison to cultureꢀdependent variation
which is inherent in analysis of CFEs.
As additional confirmation of the efficacy of our screen for the
detection of nitrilase activity, a comparison was made between
levels of ammonia detected by the established Nessler method
6
7
.
.
F. Effenberger and S. Oβwald, Synthesis (Stuttg)., 2001, 1866–1872.
D. E. Robertson, J. A. Chaplin, G. DeSantis, M. Podar, M. Madden,
E. Chi, T. Richardson, A. Milan, M. Miller, D. P. Weiner, K. Wong,
J. McQuaid, B. Farwell, L. A. Preston, X. Tan, M. A. Snead, M.
Keller, E. Mathur, P. L. Kretz, M. J. Burk, and J. M. Short, Appl.
Environ. Microbiol., 2004, 70, 2429–2436.
and by OPA reagent. Solutions of four active CFEs (entries 3a
,
8
.
S. van Pelt, M. Zhang, L. G. Otten, J. Holt, D. Y. Sorokin, F. van
Rantwijk, G. W. Black, J. J. Perry, and R. A. Sheldon, Org. Biomol.
5
,
19 and 22) were rescreened against twentyꢀnine structurally
diverse substrates and the ammonia concentrations determined
by both Nessler’s reagent (Sigma Aldrich) and OPA reagent.††
Chem., 2011, 9, 3011–3019.
Comparison of the concentrations of ammonia for 23 of these 9. L. Martínková and V. Křen, Curr. Opin. Chem. Biol., 2010, 14, 130–
substrates shows a significant correlation (Spearman rank
correlation, ρ(23) = 0.995, 0.910, 0.603 and 0.962 respectively;
P ≤ 0.002). Comparison was not possible for a further six
substrates as a precipitate was formed in the Nessler solutions
with some CFEs.
1
37.
0. J.ꢀS. Gong, Z.ꢀM. Lu, H. Li, J.ꢀS. Shi, Z.ꢀM. Zhou, and Z.ꢀH. Xu,
Microb. Cell Fact., 2012, 11
1
1
.
1. G. DeSantis, K. Wong, B. Farwell, K. Chatman, Z. Zhu, G.
Tomlinson, H. Huang, X. Tan, L. Bibbs, P. Chen, K. Kretz, and M. J.
Burk, J. Am. Chem. Soc., 2003, 125, 11476–11477.
Conclusions
1
2. G. DeSantis, Z. Zhu, W. A. Greenberg, K. Wong, J. Chaplin, S. R.
Hanson, B. Farwell, L. W. Nicholson, C. L. Rand, D. P. Weiner, D.
E. Robertson, and M. J. Burk, J. Am. Chem. Soc., 2002, 124, 9024–
The use of oꢀphthalaldehyde to determine nitrilase activity of
crude cellꢀfree extracts has been demonstrated, and been used
to determine selectivity of a panel of enzymes against a wide
9
025.
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DOI: 10.1039/C C06021K
Jou4Crnal Name
1
1
1
1
1
1
1
3. A. Banerjee, P. Kaul, and U. C. Banerjee, Arch. Microbiol., 2006,
84, 407–418.
4. L. Martínková, V. Vejvoda, and V. Křen, J. Biotechnol., 2008, 133
18–326.
5. C. O’Reilly and P. D. Turner, J. Appl. Microbiol., 2003, 95, 1161–
174.
6. A. Banerjee, P. Kaul, R. Sharma, and U. C. Banerjee, J. Biomol.
Screen., 2003, , 559–565.
1
,
3
1
8
7. Y.ꢀC. He, C.ꢀL. Ma, J.ꢀH. Xu, and L. Zhou, Appl. Microbiol.
Biotechnol., 2011, 89, 817–823.
8. C. MolinsꢀLegua, S. MeseguerꢀLloret, Y. MolinerꢀMartinez, and P.
CampínsꢀFalcó, Trends Anal. Chem., 2006, 25, 282–290.
9. A. Banerjee, R. Sharma, and U. C. Banerjee, Biotechnol. Appl.
Biochem., 2003, 37, 289–293.
2
2
2
2
2
0. S. S. Simons and D. F. Johnson, J. Org. Chem., 1978, 43, 2886–2891.
1. E. Winter, Justus Liebigs Ann. Chem., 1900, 311, 353–362.
2. L. Seekles, Recl. des Trav. Chim. des PaysꢀBas, 1924, 93–102.
3. J. C. Bill and D. S. Tarbell, Org. Synth. Collect., 1963,
4. C. M. Hong and A. V Statsyuk, Org. Biomol. Chem., 2013, 11, 2932–
935.
5. E. Kulla and P. Zuman, Org. Biomol. Chem., 2008,
4, 807–808.
2
2
2
6, 3771–3780.
6. T. Do Minh, A. L. Johnson, J. E. Jones, and P. P. Senise, J. Org.
Chem., 1977, 42, 4217–4221.
4
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