Nitrilase activity screening on structurally diverse substrates: Providing biocatalytic tools for organic synthesis

Article abstract of DOI:10.1002/adsc.201201098

A high-throughput screening of candidate nitrilases against 25 structurally diverse substrates allowed us to create a wide collection of 125 experimentally validated nitrilases. The enzymes were selected by genomic approach from 700 diverse prokaryotic species and one metagenome as representative of the nitrilase family diversity. The enzymatic screening of this collection expands the biocatalytic toolbox for chemical synthesis by providing a large number of tested nitrilases with their assigned substrates. Three examples illustrate the synthetic potential of our enzyme collection. The syntheses of carboxylic acid building blocks, a β-substituted phenylpropanoic acid, a cyclic γ-keto carboxylic acid and a mononitrile monocarboxylic acid, were achieved from the corresponding nitrile substrates, using three new nitrilases (two from Sphingomonas wittichii and one from Syntrophobacter fumaroxidans). Improvements of nitrilase activities through the optimization of reaction parameters and the preparative biocatalytic synthesis are presented for these three examples. Copyright

Full text of DOI:10.1002/adsc.201201098

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DOI: 10.1002/adsc.201201098
Nitrilase Activity Screening on Structurally Diverse Substrates:
Providing Biocatalytic Tools for Organic Synthesis
Carine Vergne-Vaxelaire,a,b,c Franck Bordier,a,b,c Aurꢀlie Fossey,a,b,c
Marielle Besnard-Gonnet,a Adrien Debard,a Aline Mariage,a Virginie Pellouin,a
Alain Perret,a,b,c Jean-Louis Petit,a Mark Stam,a,b,c Marcel Salanoubat,a,b,c
Jean Weissenbach,a Vꢀronique De Berardinis,a and Anne Zaparuchaa,b,c,
CEA, DSV, IG, Genoscope, 2 rue Gaston Crꢀmieux, 91057 Evry, France
*
a
Fax : (+33)-1-6087-2514; phone: + (33)-1-6087-4578; e-mail: azaparuc@genoscope.cns.fr
CNRS-UMR8030, 2 rue Gaston Crꢀmieux, 91057 Evry, France
Universitꢀ d’Evry Val d’Essonne, boulevard FranÅois Mitterrand, 91057 Evry, France
b
c
Received: December 17, 2012; Revised: March 21, 2013; Published online: June 4, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201201098.
Abstract: A high-throughput screening of candidate propanoic acid, a cyclic g-keto carboxylic acid and
nitrilases against 25 structurally diverse substrates al- a mononitrile monocarboxylic acid, were achieved
lowed us to create a wide collection of 125 experi- from the corresponding nitrile substrates, using three
mentally validated nitrilases. The enzymes were se- new nitrilases (two from Sphingomonas wittichii and
lected by genomic approach from 700 diverse pro- one from Syntrophobacter fumaroxidans). Improve-
karyotic species and one metagenome as representa- ments of nitrilase activities through the optimization
tive of the nitrilase family diversity. The enzymatic of reaction parameters and the preparative biocata-
screening of this collection expands the biocatalytic lytic synthesis are presented for these three exam-
toolbox for chemical synthesis by providing a large ples.
number of tested nitrilases with their assigned sub-
strates. Three examples illustrate the synthetic poten-
tial of our enzyme collection. The syntheses of car- Keywords: biocatalysis; building blocks; enzyme di-
boxylic acid building blocks, a b-substituted phenyl- versity; high-throughput screening; nitrilases
Introduction
The search for new nitrilases remains relevant to pro-
vide efficient biocatalysts for synthetic applications.[3]
Since nitrilases are widespread among microorgan-
The chemical hydrolysis of nitriles in their corre-
sponding carboxylic acids often leads to unwanted by- isms, efficient mining of enzyme and sequence data-
products due to the drastic conditions required such bases is increasingly important to enrich the biocata-
as strong bases, acids or elevated temperatures. Bio- lyst catalogue.[3c] Bioinformatic approaches have to be
catalyzed hydrolysis by nitrilases has become a great combined with experimental biochemical data to pre-
environmentally benign alternative because of its effi- cisely define or confirm the enzyme conversion capa-
ciency and stereospecificity associated with the very bilities. This biochemical work is needed since the
mild conditions used.[1] Nitrilases (E.C.3.5.5) are functional annotation of a given gene is mainly infer-
a subcategory of the carbon-nitrogen hydrolase super- red from homology analysis without any experimental
family that catalyse non-peptide carbon-nitrogen evidence. Moreover, the prediction of the chemical
bond hydrolysis, with some of them also having a ni- reaction catalyzed by a gene product is generally
trile hydratase secondary activity.[2] Intensive research vague with no information regarding the substrate
on nitrilase-mediated biocatalysis has occurred in the specificity.
past decades and dozens of nitrilases have already
Combining Genoscope bioinformatic skills and its
been found and characterized.[1e] However, industries high throughput process know-how we created a cata-
and chemists still need new ones with higher activi- logue of experimentally characterized nitrilases. We
ties, wider specificities and more robust stabilities. considered this issue through two aspects. Firstly, how
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specific are the enzymes of our set? Secondly, how genes in Escherichia coli BL21, cells were lyzed and
important is the substrates range covered by the enzy- the proteins quantified. All these steps were conduct-
matic diversity? We report here the results of the ed in 96-microwell plates. These plates were next
screening of 163 candidate nitrilases against 25 struc- screened against 25 nitriles (commercially available or
turally diverse nitriles and illustrate synthetic utility synthesized in our laboratory, see Experimental Sec-
through three examples.
tion) chosen to cover a wide structural diversity: satu-
rated (1–5), unsaturated (6–11), arylaceto- (12–16), a-
hydroxylated (17–19), b-hydroxylated (20–22) and b-
aminated (23–25) nitriles (Figure 1).
Results and Discussion
Protein and Substrate Selection
High Throughput Activity Screening of Candidate
Sequences coding for experimentally confirmed nitri- Nitrilases on Selected Nitriles
lases were collected and used for similarity sequence
analyses using UniprotKB and Genoscope databases Nitrilase Activity Measurement
(see Experimental Section). Candidate nitrilases were
then selected according to their genome availability The nitrilase assay in high throughput screening is
in the Genoscope strain collection (700 prokaryote generally based on NH3 detection, NH3 being stoi-
species) and in an in-house wastewater treatment chiometrically produced along with the carboxylic
plant metagenomic collection of sequences (Cloaca acid in the reaction. Different fluorometric or pH in-
maxima).[4] Around 290 candidate nitrilases were se- dicators methods are available.[5] Nevertheless, we de-
lected as representative of the nitrilase diversity. cided to monitor the catalytic activity by an enzymatic
Among them, only 12 proteins were annotated as “ni- spectrophotometric coupled assay using glutamate de-
trilases”. Most of the selected enzymes were annotat- hydrogenase (GDH) (Scheme 1).
ed as carbon-nitrogen hydrolases or only hydrolases.
In this reaction, the ammonia liberated by the ni-
This set also included 25 enzymes annotated as “con- trile hydrolysis is used as co-substrate, in the presence
served hypothetical protein”. From the 290 candi- of an excess of a-ketoglutaric acid, by glutamate de-
dates, 163 genes were successfully cloned with a hexa- hydrogenase. We monitored the oxidation of NADH
histidine tag for further purification purposes. Cloning that reflects indirectly NH3 consumption at 340 nm by
failures were mostly due to the very high GC content UV spectrophotometry.[6] This robust and sensitive
(>70%) of many genes, preventing efficient PCR am- assay (down to 25 mM of NH3 could be quantified in
plifications. After overexpression of recombinant this 96-microwell plates assay; not shown) being well
Figure 1. Substrates used for the enzymatic activity screening.
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
Scheme 1. Enzymatic spectrophotometric coupled assay using glutamate dehydrogenase.
adapted to high throughput screening led us to rapid- a weak one (Figure 2 and Supporting Information Fig-
ly index nitrilases relative to their substrate specifici- ure S1). 21 proteins showed no activity even if a pro-
ties. To the best of our knowledge, this assay has duction of soluble enzyme was observed on SDS-
never been used for the monitoring of nitrilase activi- PAGE. Some nitrilases seemed to be substrate specif-
ty. The screening was thus realized in 96-microwell ic such as NIT61 for pentanenitrile (1). The majority
plates in the presence of 10% MeOH (see Experi- were promiscuous, hydrolysing a broad substrate
mental Section). The use of MeOH enabled substrate scope, mainly nitriles bearing an aromatic moiety
solubilization. This solvent is commonly used in nitri- (77% of the active proteins), saturated nitriles (60%)
lase activity screening in the range of 5–10%.[3a,b] It and unsaturated nitriles (50%). It is worth noting that
was reported to induce no (or only slight) diminution around 60% of the enzyme set were active towards at
of nitrilase activity. The activity, expressed in arbitrary least one compound representative of both substrate
units, was obtained after subtraction of a blank reac- groups bearing or not an aromatic moiety, illustrating
tion without substrate and threshold settings the large nitrilase promiscuity (Supporting Informa-
(Figure 2). Due to enzyme overexpression variations tion Figure S2). Two very promiscuous nitrilases
in each cell lysate, the relative activity toward various showed a significant activity on at least one substrate
substrates can be compared on a same cell lysate but by nitrile group: NIT93 and NIT278. Around 35% of
not between cell lysates.
the nitrilases showed an activity on a-hydroxylated or
b-hydroxylated nitriles, and 16% exhibited both activ-
ities. Only 8% showed an activity on b-aminated ni-
triles and no significant nitrilase activity could be de-
tected for the 3-amino-3-phenylpropanenitrile (25)
Activity Screening Results
Preliminary results revealed that 125 out of the 163 (see Supporting Information, Figure S2 and S3).
tested proteins exhibited a nitrilase activity, even These characteristics are promising results for syn-
Figure 2. Activity screening results of 125 nitrilases against 25 nitriles: the activity is expressed in an arbitrary unit related to
the initial slope of disappearance of NADH observed at 340 nm.
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Table 1. Catalytic activity of the 27 purified nitrilases towards
Methodological studies have been conducted to opti-
mize the conversion rate and the stereospecificity.
Some reaction parameters (pH, co-solvent, tempera-
ture) have indeed already been reported to have a no-
table influence.[7]
the 25 nitriles.[a]
Synthesis of 4-Methoxy-4-oxo-3-phenylbutanoic
Acid[8a]
The screening results showed many highly active hits
towards the 3-phenylpropionitrile (3), which was al-
ready reported to be substrate of nitrilases.[3e,9,10] We
decided to assay these enzymes against a structurally
related b-ester nitrile, the methyl 3-cyano-2-phenyl-
propanoate (26) since the organic synthesis of the re-
sulting carboxylic acid 27 containing two similar func-
tions in the relative b position is highly difficult, and
only few examples (requiring toxic reagents) are re-
ported.[11] The enantiomerically enriched b-methyl
ester carboxylic acid 27 is a valuable precursor of the
quinolinone skeleton found in various biologically
active compounds (Scheme 2).[12] No methyl 3-cyano-
[a]
Coded results: &=high activity (ꢀ1 mM of substrate
converted in 4 h); &=moderate activity (<1 mM of sub-
strate converted in 4 h); &=no activity.
thetic applications starting from structurally diverse 2-phenylpropanoate nitrilase has been reported to the
nitriles.
best of our knowledge and a biocatalytic transforma-
To validate our screening conducted on cell lysates, tion would be an efficient alternative.
for each substrate, a few enzymes out of those exhib-
The seven enzymes exhibiting the highest apparent
iting the best activities were selected (37 in all), puri- activity towards nitrile 3 in our screening (Table 1)
fied by nickel affinity chromatography (from a 50-mL NIT28, NIT131, NIT158, NIT161, NIT188, NIT191
culture), and tested according to the same GDH and NIT278, were individually purified on a larger
assay.
scale (from 250-mL culture) by nickel affinity chro-
The results (Table 1) were mostly consistent with matography. The purified nitrilases were then tested
the previous ones as 27 nitrilase activities were con- on the methyl 3-cyano-2-phenylpropanoate (26) readi-
firmed using purified enzymes, attesting that we have ly accessible by chemical synthesis.[13] Three enzymes
now in hand a broad collection of nitrilases. No activi- proved to be active towards ester 26, NIT28, NIT158
ty could be detected for the 10 remaining enzymes, and NIT191, (Table 2) and one of the three was stud-
suggesting a loss of activity upon purification or a low ied in greater detail because of its stereospecificity,
purification yield. Classification of the screening re- NIT28 from Sphingomonas wittichii (A5V4Y8).
sults according to the substrates showed that the
structural diversity was well covered by our nitrilase
set as only one compound, the 3-aminopentanenitrile Study of Sphingomonas wittichii Nitrilase (NIT28)
(24), was not hydrolyzed to its corresponding acid. It Activity
is worthy to note that the 3-amino-3-phenylpropane-
nitrile (25) was found to be a substrate of the purified The main parameters influencing the rate of the bio-
NIT278 although no activity was detected on cell catalytic reaction were studied to optimize the prepa-
lysate.
Biocatalytic Synthesis of Key Organic Synthons with
Discovered Nitrilases
We wanted to illustrate the potential of our collection
of nitrilases as a toolbox dedicated for the organic
chemists. In particular, we intended to demonstrate
that it could be useful for the synthesis of building
blocks difficult to synthetize using conventional or-
ganic strategies. Three examples are developed here. phenylpropanoate (26).
Scheme 2. Biocatalytic transformation of methyl 3-cyano-2-
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
Table 2. Activity of selected enzymes on studied nitriles.[a]
of the unwanted amides from starting nitriles was de-
pendent on the reaction conditions (vide infra). The
influence of the buffer molarity on the nitrilase rela-
tive activity was studied between 10 and 200 mM in
potassium phosphate buffer at pH 7.3 (Figure 3C).
The increase in molarity led to an improvement of up
to 20% of the relative activity; nevertheless a 30 mM
molarity was chosen for the preparative scale reaction
to avoid the presence of too many salts that would
have to be removed during the purification steps. The
optimum temperature appeared to be 408C (Fig-
ure 3D) but formation of amide was observed for
temperatures above 358C (data not shown). Conse-
quently 308C was chosen for the preparative scale re-
action to avoid the formation of this unwanted by-
product. The effect of various co-solvents (ethylene
glycol, DMSO, MeOH, MeCN, THF, toluene and
CHCl3) in 5% volume was studied (Figure 3E) as pre-
vious work showed a potential influence on conver-
sion and enantiospecificity.[7b] Unfortunately, no im-
provement on the enantiomeric excess was observed
(data not shown). We even noticed a slight diminution
of the enantiospecificity in the presence of 5% THF.
The highest activity was measured with ethylene
glycol and DMSO compared to MeOH, and no activi-
ty was detected with toluene and CHCl3. Nevertheless
MeOH was chosen for the preparative scale reaction
for its easier removal. It is worth noting that replacing
the methyl group of the substrate ester moiety by
a more sterically hindered substituent like ethyl, iso-
propyl or benzyl did not improve the enantiospecifici-
ty and dramatically decreased the activity (data not
[a]
Coded results: &=high activity (ꢀ1 mM of substrate
converted in 4 h); &=moderate activity (<1 mM of sub-
strate converted in 4 h); &=no activity; &=not tested.
The activity was tested by GDH assay on purified en-
zymes and analyzed also by HPLC for compounds 26,
28a and 28b (except for *). Activity towards compounds
2, 10 and 30 was tested on crude cell lysates.
rative scale reaction in terms of conversion and enan- shown).
tiospecificity: pH, buffer nature and molarity, temper-
ature and nature of the co-solvent used. The conver-
sion and the enantiospecificity were studied by HPLC Preparative Scale Reaction
by comparison with synthetic standards (see Experi-
mental Section for HPLC conditions and synthesis). Under optimized reaction conditions (10 mM of sub-
The enantiospecificity was not clearly affected either strate with 5% MeOH in potassium phosphate buffer
by the pH, or the buffer nature and molarity, the tem- 30 mM pH 7.3 at 308C), the reaction was performed
perature or the nature of the co-solvent (data not on a preparative scale (47 mg of nitrile with 0.37 mg
shown). The effects of these parameters on the con- of enzyme) and the (R)-4-methoxy-4-oxo-3-phenylbu-
version are reported in Figure 3.
tanoic acid (27) was isolated in 96Æ3% yield (based
S. wittichii nitrilase (0.05 mgmLÀ1 of purified on 50% conversion) and 46Æ2% of enantiomeric
enzyme (A5V4Y8), see Experimental Section) con- excess after 7 h30 of incubation time (see Experimen-
verted 50% of 5 mM 3-cyano-2-phenylpropanoate tal Section). The purification protocol also led to the
(26) with a preference for the (R) enantiomer result- isolation of recovered nitrile (93%; ee=42Æ4%).
ing in an enantiomeric excess of 50% (Figure 3A). No
excess-substrate inhibition was observed for substrate
concentrations up to 10 mM and higher concentra- Synthesis of 3-Oxocyclopentanecarboxylic Acid[8b]
tions induced solubility problems. The enzyme
showed the highest activity at pH 7–8, displaying We next focused on the hydrolysis of cyclic g-keto ni-
a similar activity in potassium phosphate as well as in triles 28,[14] the corresponding chiral cyclic g-keto
Tris.HCl buffer (Figure 3B). No pH effect on the acids 29 being valuable building blocks for the synthe-
enantiomeric excess was noticed (data not shown). sis of fine chemicals (Scheme 3).
Formation of the amide was observed at pH higher
Only a few analogues are described in the literature
than 9. More generally, we noted that the appearance to be substrates of nitrilases, particularly 1-cyanocy-
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Figure 3. Effect of various parameters on the conversion of 5 mM methyl 3-cyano-2-phenylpropanoate (26) and/or enantio-
specificity by NIT28. (A) Evolution of the conversion ^ and enantiospecificity &. Reaction carried out at 308C in 100 mM
potassium phosphate buffer pH 7.3 and 5% MeOH. (B) Effect of the pH on the nitrilase relative activity. Reactions carried
out for 10 min at 308C in potassium phosphate buffer 100 mM ^ and Tris·HCl 100 mM & with 5% MeOH. (C) Effect of
the buffer molarity on the nitrilase relative activity. Reactions carried out at 308C for 3 h with 5% MeOH in potassium phos-
phate buffer pH 7.3 at various molarities. (D) Effect of the temperature on the nitrilase relative activity. Reactions carried
out for 30 min in potassium phosphate buffer 100 mM pH 7.3 and 5% MeOH. (E) Effect of the co-solvent on the nitrilase
relative activity. Reactions carried out at 308C for 35 min in potassium phosphate buffer 100 mm pH 7.3 and 5% of various
co-solvents.
cloalkaneacetonitrile (precursor of gabapentin and
analogues) which is regioselectively hydrolyzed by the
nitrilase Q89GE3 from Bradyrhizobium japonicum
strain USDA110,[15] carbocyclic g-amino nitriles (pre-
cursors of GABA analogues) by commercial nitrilas-
es,[16] and 3-(3-oxocyclohexyl)propanenitrile by Rho-
Scheme 3. Biocatalytic hydrolysis of 3-oxocyclopentane- and
3-oxocyclohexanecarbonitriles 28.
dococcus sp. SP361.[17] The cyclic g-keto nitriles 28a
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
Figure 4. Effect of various parameters on the conversion of 5 mm 3-oxocyclopentanecarbonitrile (28a) and/or enantiospecific-
ity by NIT278. (A) Evolution of the conversion ^ and enantiospecificity &. Reaction carried out at 308C in 100 mM potas-
sium phosphate buffer pH 7.3. (B) Effect of the pH on the nitrilase relative activity. Reactions carried out for 2 h30 min at
308C in potassium phosphate buffer 100 mM ^ and Tris·HCl 100 mM &. (C) Effect of the buffer molarity on the nitrilase
relative activity. Reactions carried out for 2 h30 min at 308C in potassium phosphate buffer pH 7.3. (D) Effect of the tem-
perature on the nitrilase relative activity. Reactions carried out for 2 h30 min in potassium phosphate buffer 100 mM pH 7.3.
(E) Effect of the co-solvent on the nitrilase relative activity. Reactions carried out at 308C for 2 h30 min in potassium phos-
phate buffer 100 mM pH 7.3 and 5% of various co-solvents.
and/or 28b were tested against the 37 purified nitrilas- dans (A0LKP2), was therefore studied in greater de-
es; five enzymes (NIT28, NIT158, NIT161, NIT191 tails on the 3-oxocyclopentanecarbonitrile (28a).
and NIT278) among the nine hits obtained were fur-
ther analyzed by HPLC to determine the best candi-
date combining good activity (Table 2) and enantio- Study of Syntrophobacter fumaroxidans Nitrilase
specificity (data not shown).[18] As the most promising (NIT278) Activity
results were found on the five-membered ring 28a, we
decided to focus our study on this substrate. One of The main parameters influencing the biocatalytic re-
the best hits, NIT278 from Syntrophobacter fumaroxi- action were studied in the same way as for NIT28 ac-
tivity (vide supra). The conversion and the enantio-
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specificity were studied by HPLC by comparison with Synthesis of 2-[(Cyanomethyl)amino]acetic Acid[8c]
a synthetic standard (see Experimental Section for
HPLC conditions and synthesis). The enantiospecifici- Finally, the biotransformation of dinitriles attracted
ty was not clearly affected either by the pH, or the also our attention due to the enzymeꢂs ability to de-
buffer nature and molarity, the temperature or the symmetrize the substrates by producing monoacid
nature of the co-solvent (data not shown). The effects mononitrile products.[19] From our initial candidate
of these parameters on the conversion are reported in enzymes set, 15 proteins proved to be active towards
Figure 4.
the common dinitriles glutaronitrile (2) and/or fumar-
S. fumaroxidans (A0LKP2) nitrilase (0.1 mgmLÀ1 onitrile (10) (see Table 2 and Supporting Information,
of purified enzyme, see Experimental Section) con- Figure S1). The 15 crude cell lysates were therefore
verted 50% of 5 mM 3-oxocyclopentanecarbonitrile screened against six different commercially available
(28a) with a preference for the (S) enantiomer result- dinitriles not present in the first substrate set: malo-
ing in an enantiomeric excess of 50% (Figure 4A). A nonitrile, succinonitrile, adiponitrile, suberonitrile,
slight excess-substrate inhibition was observed for trans-3-hexenedinitrile and iminodiacetonitrile. Each
a concentration above 30 mM. The enzyme showed of these compounds revealed to be a substrate of at
the highest activity at pH 7–8 (Figure 4B). We noticed least one of the tested nitrilases. We decided to focus
a clear effect of the buffer nature on nitrilase activity, on dinitrile 30 because, to the best of our knowledge,
the maximum activity being obtained in potassium no enzyme was described to hydrolyze only one of its
phosphate buffer rather than in Tris·HCl. No pH two nitrile functions (Scheme 4), whereas nitrilases
effect on the enantiomeric excess was observed. As were proved to hydrolyse selectively the other tested
seen for the NIT28 enzyme (vide supra), an increase dinitriles: B. japonicum strain USDA110,[15] Synecho-
in the buffer concentration led to an improvement of cystis sp. strain PCC 6803,[10] Rhodococcus sp., a modi-
up to 2-fold when compared to the activity measured fied nitrilase from Alcaligenes faecalis,[20] Nit1 from
at 10 mM (Figure 4C). A molarity of 80 mM was a metagenome library.[21] Whole cells of A. faecalis
chosen for the preparative scale reaction, as the ZJB-09133 were used to produce iminodiacetic acid
enzyme showed a strong activity in these conditions. (31) on a preparative scale.[22] In organic synthesis,
As for NIT28, the optimum temperature appeared to formation of the amino acid 32 was reported starting
be around 408C (Figure 4D). A temperature of 378C from glycine but as a non-isolated intermediate.[23]
was therefore chosen for the preparative scale reac-
Seven nitrilases catalyzed the hydrolysis of imino-
tion. The presence of 5% of various co-solvents did diacetonitrile (30) (Table 2). To address the regiose-
not improve either the enantiospecificity (data not lectivity issue, these seven enzymes were purified and
shown) or the conversion rate (Figure 4E). The prepa- further studied by HPLC after derivatization of the
rative scale reaction was then conducted without any reaction mixture with 1-fluoro-2,4-dinitrobenzene
co-solvent, the substrate being soluble at 30 mM. This (FDNB) and comparison with synthetic standards
enzyme showed also a reduced activity on the six- (see Experimental Section). Nitrilases NIT28,
membered ring analogue 28b, along with a weaker NIT158, NIT188 and NIT206 exhibited a mononitrile
stereospecificity. It is worth noting that the amide for- hydrolase activity whereas the nitrilases NIT161,
mation was predominantly observed with NIT161 NIT191 and NIT278 proved to be almost exclusively
(from Genoscope Cloaca Maxima metagenome) for dinitrilases (Figure 5). Being easily overproduced,
both 28a and 28b and that the stereopreference was NIT158 from S. wittichii (A5VE02) was chosen for
determined by the ring size (data not shown).
reaction optimization and scale-up.
The reaction parameters were optimized for a prep-
arative scale reaction (Figure 6) as previously de-
scribed for NIT28. The conversion was analyzed by
HPLC (see Experimental Section for HPLC condi-
Preparative Scale Reaction
Using the optimized conditions, the reaction with sub- tions).
strate 28a was conducted on a 30-mg scale (30 mM
28a, potassium phosphate buffer 80 mM pH 7.3, 378C,
0.4 mg of purified enzyme) and after a 48 h incuba-
tion, (S)-29a was isolated in 74Æ5% yield (based on
50% conversion) and 48Æ2% of enantiomeric excess
(see Experimental Section).
Scheme 4. Biocatalytic hydrolysis of the iminodiacetonitrile
(30).
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
A slight excess-substrate inhibition was observed
above 30 mM (data not shown). The enzyme showed
the highest activity at pH 7–9 in potassium phosphate
buffer (Figure 6), but in reactions conducted at a pH
above 8.5, the formation of some diacid was observed.
These data indicated that the best conditions for
a preparative scale reaction should be chosen at a pH
close to 7. Using Tris·HCl as the buffer decreased no-
tably the activity. To avoid the presence of too many
salts that would have to be removed during the purifi-
cation steps, a buffer molarity of 30 mM was chosen
for the preparative scale reaction, as the enzyme
showed a similar activity in these conditions. The tem-
perature profile showed an optimum activity at 35–
408C (Figure 6C). The presence of 5% of various co-
Figure 5. Time-dependent studies on the hydrolysis of imi-
nodiacetonitrile 30 (10 mM) by purified nitrilases in potassi- solvents significantly pulled down the conversion,
um phosphate buffer 100 mM pH 7.3, 5% MeOH at 308C.
The reaction times were 3, 24 and 48 h (except for NIT191,
3 and 24 h).
even methanol which usually does not affect the activ-
ity (vide supra) (Figure 6D).
Figure 6. Effect of various parameters on the conversion of 10 mM of iminodiacetonitrile (30) by NIT158. (A) Evolution of
the conversion ^. Reaction carried out at 308C in 100 mM potassium phosphate buffer pH 7.3. (B) Effect of the pH on the
nitrilase relative activity. Reactions carried out for 4 h30 min at 308C in potassium phosphate buffer 100 mM ^ and
Tris·HCl 100 mM & (derivatization: conditions 2). (C) Effect of the temperature on the nitrilase relative activity. Reactions
carried out for 4 h30 min in potassium phosphate buffer 100 mM pH 7.3 (derivatization: conditions 1). (D) Effect of the co-
solvent on the nitrilase relative activity. Reactions carried out at 308C for 4 h30 min in potassium phosphate buffer 100 mM
pH 7.3 and 5% of various co-solvents (derivatization: conditions 1).
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Preparative Scale Reaction
(Table 3). For example, NIT28 shows an identity of
61% with the nitrilase of Burkholderia xenovorans,[3c]
The optimized reaction conditions (30 mM of dinitrile NIT158 and NIT278 have 46% and 64% identity, re-
30, potassium phosphate buffer 30 mM pH 7.0 without spectively, with the nitrilases of B. japonicum.[15,3g]
co-solvent, 378C, 24 h incubation time) were applied More generally, about 60% of our nitrilases have less
to produce 25 mg of 2-[(cyanomethyl)amino]acetic than 40% identity with the known nitrilases (Table 3).
acid (32) (73% isolated yield) from crude cell lysate Most of the time the nitrilase of Pyrococcus abyssi is
(see Experimental Section). As purified enzyme and their best known homologue (Table 3).[25] As shown in
crude cell lysate both led to similar results, the scale- the phylogenetic tree (Figure 7), these nitrilases (19
up was carried out with a crude cell lysate for a simpli- over 31) seem to form a distant group. Moreover,
fied procedure.
these nitrilases are shorter (~260 amino acids versus
~315 to 360 amino acids) than the others, including
those of the eukaryotic Arabidopsis thaliana. So far,
only one nitrilase from archaebacteria was character-
ized (Q9UYV8 from the Euryarchae P. abyssi), and
Sequence Analysis
In order to evaluate the sequence novelty of our 31 three new archaebacterial nitrilases are reported here
purified nitrilases, we compared their amino acid se- (NIT265, NIT141 and NIT228).[25] Interestingly,
quences to the ones of 20 prokaryotic nitrilases puri- NIT228 from Sulfolobus tokodaii is the first studied
fied and experimentally characterized, and for which nitrilase from the Crenarchaeota branch of archae-
a protein sequence was associated in a publication bacteria. Since enzymes from archaebacteria are very
Table 3. Nitrilase best hits and percentage of identity with published nitrilases experimentally validated and related to an
UniprotKB entry.
NIT
Uniprot KB entry
Identity[a]
Best[b] homologue
Organism[b]
Ref.
NIT08
NIT15
NIT18
NIT22
NIT24
NIT26
NIT28
NIT32
A8YU05
A6GDG9
A5VIE2
A8FQL4
Q13FH1
Q7MB65
A5V4Y8
Q47S40
A2TUX6
A7HS79
Q5NN79
Q5LWM4
F9VNT2
F9UU19
A0KFB4
A2STE2
A1ALZ7
A5VE02
32%
30%
33%
29%
41%
63%
61%
32%
34%
34%
55%
28%
33%
32%
35%
27%
34%
46%
52%
31%
31%
68%
100%
34%
54%
31%
31%
64%
68%
46%
28%
Q9UYV8
Q9UYV8
Q9UYV8
E1A0Z9
Q9UYV8
Q55949
Pyrococcus abyssi
Pyrococcus abyssi
Pyrococcus abyssi
uncultivated bacterium
Pyrococcus abyssi
Synechocystis sp.
[25]
[25]
[25]
[21]
[25]
[24]
[3c]
[25]
[3a]
[25]
[34]
[25]
[25]
[25]
[25]
[25]
[25]
[15]
[3g]
[25]
[25]
[24]
[3c]
[25]
[24]
[25]
[25]
[3g]
[24]
[25]
[25]
Q13VV3
Q9UYV8
Q6RWK4
Q9UYV8
Q4KCl8
Q9UYV8
F9VNT2
F9UU19
Q9UYV8
Q9UYV8
Q9UYV8
Q89GE3
Q89PT3
Q9UYV8
Q9UYV8
Q55949
Q13VV3
Q9UYV8
Q55949
F9VNV6
Q9UYV8
Q89PT3
Q55949
Burkholderia xenovorans
Pyrococcus abyssi
uncultivated bacterium
Pyrococcus abyssi
Pseudomonas fluorescens
Pyrococcus abyssi
Pyrococcus abyssi
Pyrococcus abyssi
Pyrococcus abyssi
Pyrococcus abyssi
Pyrococcus abyssi
Bradyrhizobium japonicum
Bradyrhizobium japonicum
Pyrococcus abyssi
Pyrococcus abyssi
Synechocystis sp.
Burkholderia xenovorans
Pyrococcus abyssi
Synechocystis sp.
Pyrococcus abyssi
Pyrococcus abyssi
Bradyrhizobium japonicum
Synechocystis sp.
NIT38
NIT56
NIT66
NIT122
NIT125
NIT128
NIT131
NIT141
NIT144
NIT158
NIT161
NIT171
NIT180
NIT188
NIT191
NIT205
NIT206
NIT228
NIT265
NIT278
NIT280
NIT282
NIT283
Q21FT6
A8F9S6
B2JQY2
Q13VV3
B1MI91
B2JMC7
F9VNV6
Q9HIW8
A0LKP2
A8IGC7
B5YA14
B1MBK5
Q9UYV8
Q9UYV8
Pyrococcus abyssi
Pyrococcus abyssi
[a]
Percentage of identity with the best homologue.
Best published homologue with experimental data.
[b]
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
Figure 7. Phylogenetic analysis of nitrilase sequences. The tree was done with the 31 nitrilases experimentally validated in
this work and 23 previously published nitrilases experimentally validated and associated with a protein sequence. All the ni-
trilase UniprotKB identifiers are reported in the tree. The references for known nitrilases are into brackets. Bootstrap
values (in red) are given when the score is up to 80%. Activity of selected enzymes on studied nitriles are symbolized by
colors. Coded results: &=high activity (ꢀ1 mM of substrate converted in 4 h); &=moderate activity (<1 mm of substrate
converted in 4 h); &=no activity; &=not tested.
promising biocatalysts for synthetic chemistry and in- abyssi, seem, under our conditions, to be specific for
dustries due to their thermal stability, their capabili- aliphatic nitriles including b-hydroxylated nitriles.
ties are under investigation in the laboratory. We ana-
lyzed our results regarding a possible connection be-
tween sequence and substrate specificity. The result Conclusions
was not conclusive as it was not possible to highlight
a specific sequence–activity relationship due to the The present study demonstrates the efficiency of our
large substrate promiscuity of the nitrilases. Neverthe- approach of combining sequence data analyses with
less, the nitrilases from A. faecalis and Pseudomonas a high throughput experimental activity screening, to
fluorescens, known to have arylacetonitrilase activi- target the identification of new nitrilases. We report
ties, are close in the phylogenetic tree to NIT28 and here the set-up of a sensitive generic enzymatic assay
NIT191. It is worth noting that these latter ones are for the detection of nitrilase activity designed for high
also active towards the arylacetonitrile substrates.[22,9] throughput screening. We created a collection of 125
Moreover, the 19 nitrilases from the sub-group of P. experimentally characterized nitrilases for the bio-
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FULL PAPERS
number of candidates, protein sequences were clusterized
(80% of similarities) using traditional all-against-all normal-
ized BLASTP scores calculated by the LASSAP suite and
a single-linkage algorithm. A representative candidate was
then chosen, one by cluster, if its corresponding bacterial
strain was available in the Genoscope genomic collection.
All the sequences too divergent to be included in the clus-
tering were chosen if the corresponding genome was avail-
able in the Genoscope genomic collection. Missing strains
were purchased from DSMZ when available.
transformation of structurally diverse nitriles (saturat-
ed, unsaturated, arylaceto-, a-hydroxylated, b-hy-
droxylated and b-aminated nitriles). Only 12 (9.6%)
of these enzymes were previously annotated as pre-
dicted nitrilases, 21 (17%) were missannotated as ami-
dohydrolases and most of them were only annotated
as putative carbon-nitrogen hydrolases. Out of the
125 studied nitrilases, 31 were purified and character-
ized for substrate specificity. The availability of these
new enzymes with defined nitrilase activities opens
the way to improve the catalytic properties by muta-
genesis for biocatalytic applications and for metabolic
engineering. Such a collection will be a very useful re-
source for further exploration of the conversion of di-
verse nitrile derivatives and provide key tools for or-
ganic chemists. The biocatalytic potential of our col-
lection is illustrated here through the synthesis of
three carboxylic acid building blocks: a b-substituted
phenylpropanoic acid, a cyclic g-ketocarboxylic acid
and a mononitrile monocarboxylic acid. Investigations
are underway to integrate these nitrilases in enzymat-
ic cascades.
Cloning and Candidate Enzyme Production
All steps from primers purchase to biochemical assays were
performed in 96 micro-wells. The choice of the primers was
based on the Primer3 program.[34] Oligonucleotides were
from Sigma-Genosys. Specific extensions are added to the
primers for cloning into our plasmid pET22b(+) (Novagen)
modified for ligation independent cloning.[35] The forward
primers introduced a hexahistidine sequence in the proteins
after the initial methionine for purification purposes. Plas-
mid were then introduced into E. coli BL21ACTHNURGTNE(NUG DE3) plysE
strains (Invitrogen) for over-expression. Cell cultures, iso-
propyl b-d-thiogalactopyranoside (IPTG) induction for pro-
tein production and cell extracts were then conducted as
previously reported.[6] For the production of the enzymes
used for the initial high throughput screening, an adaption
for 96-microwell plate conditions was needed. Briefly, a 1.6-
mL cell culture was induced for recombinant protein pro-
duction in each well. After centrifugation, cells were sus-
pended in 300 mL of 50 mM potassium phosphate buffer
pH 7.3 containing 50 mM NaCl, 10% glycerol, 1 mM Pefa-
bloc SC and 0.2 mL LysonaseTM bioprocessing reagent
(Merck), and sonicated using an ultrasonic bath (Bronson
2510 Ultrasonic Cleaner). After centrifugation to clarify the
cell extract, the supernatants were analyzed by sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) to check for recombinant protein production. Pro-
tein concentrations were determined by the Bradford
method with bovine serum albumin as the standard (Bio-
Rad). The samples were stored at À808C.
Experimental Section
Chemicals, Strains
All reagents were purchased from commercial sources and
used without additional purification. Valeronitrile (1), gluta-
ronitrile (2), 3-phenylpropanenitrile (3), isobutyronitrile (4),
acetonitrile (5), benzonitrile (6), terephthalonitrile (7), 2-cy-
anopyridine (8), 2-pentenenitrile (9), fumaronitrile (10),
trans-cinnamonitrile (11), phenylacetonitrile (12), (Æ)-2-(4’-
isobutylphenyl)propionitrile (13), indole-3-acetonitrile (14),
2-thiopheneacetonitrile (15), a-phenylcinnamonitrile (16),
glycolonitrile (17), mandelonitrile (18), 2-hydroxypentaneni-
trile (19), 3-hydroxypropanenitrile (20) and 3-aminoprop-
ACHTUNGTRENNUNGanenitrile (23) were purchased from Sigma–Aldrich. NMR
spectra were recorded on a Bruker 300 MHz spectrometer
(Evry University, France). Chemical shifts (expressed in
ppm) of 1H and 13C NMR spectra were referenced to the
solvent peaks d(H)=7.24 and d(C)=77.2 for CDCl3, d(H)=
2.50 and d(C)=39.5 for DMSO-d6. Thin-layer chromatogra-
phy was performed with aluminium-backed sheets with
silica gel 60 F254 (Merck). Column chromatography was per-
formed on a CombiFlashꢃ Companion using RediSepꢃ Rf
or GraceResolv Silica cartridges. HPLC analyses were per-
formed on a Waters model 2795 liquid chromatograph pro-
vided with a Waters model 996 Photodiode Array Detector.
Purification of Enzymes
The enzymes were purified by loading the crude extract
onto a Ni-NTA column (QIAGEN), according to the manu-
facturerꢂs instructions. Large scale purification of A5V4Y8,
A0LKP2, A5VE02 were done by nickel affinity purifications
as previously reported.[6] Protein concentrations were deter-
mined by the Bradford method. The samples were analyzed
by SDS-PAGE using the Invitrogen NuPage system. The pu-
rified proteins were stored at À808C.
High Throughput Enzymatic Assay
Choice of Candidate Enzymes
The nitrilase reactions were performed in 100 mL containing
1 mM DTT, 5 mL cell lysate (0.03 to 0.1 mgmLÀ1 of total
proteins), 10 mM substrate in 10% MeOH at pH 7.5 (0.1M
potassium phosphate buffer pH 7.5) at 308C for 4 h. The re-
actions were stopped with 1% TFA and neutralized with
5M K2CO3. 100 mL of a mixture containing 300 mM NADH,
100 mM ADP, 10 mM a-ketoglutaric acid, and 3.15
unitsmLÀ1 of glutamate dehydrogenase, in 0.1M potassium
A set of 34 nitrilases experimentally characterized according
to UniprotKB was built up. It was used for protein-versus-
protein alignments, using the BL2 option (BLAST allowing
gaps) and a BLOSUM62 score matrix against UniprotKB.
Similarities restricted to those presenting more than 30%
identity over at least 80% of the length of the smaller of
two compared sequences, were kept. To minimize the
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Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
phosphate buffer pH 7.5 were next added. The oxidation of
NADH could then be monitored to evaluate the release of
NH3, as previously reported.[6] Chemicals and glutamate de-
hydrogenase were purchased from Sigma–Aldrich. All these
reactions were conducted at 258C in 96-microwell plates in
a final volume of 200 mL using a SpectraMax Plus384 ab-
sorbance microplate reader (Molecular Devices).
ous phase was extracted with dichloromethane (3ꢄ10 mL)
and the combined organic layers were dried over magnesi-
um sulfate, filtered and concentrated to dryness under re-
duced pressure to obtain the mesylate which was used in the
next step without further purification; yield: 1.40 g.
To a suspension of the mesylate (5.3 mmol, 1 equiv.,
1.40 g) in dimethyl sulfoxide (28 mL) was added potassium
cyanide (14.0 mmol, 2.6 equiv., 908 mg). The mixture was
vigorously stirred for 18 h at 408C and then water (50 mL)
and diethyl ether (25 mL) were added to stop the reaction.
The aqueous phase was extracted with diethyl ether (3ꢄ
30 mL) and the combined organic layers were washed with
brine (2ꢄ30 mL), dried over magnesium sulfate, filtered and
concentrated to dryness under reduced pressure. To a part
of the residue (0.99 mmol, 1 equiv., 196 mg) dissolved in
dried dichloromethane (2 mL) were successively added trie-
thylsilane (2.47 mmol, 2.5 equiv., 393 mL) and trifluoroacetic
acid (12.8 mmol, 13 equiv., 951 mL) under an inert atmos-
phere at room temperature. The mixture was stirred for
15 min and then concentrated to dryness under reduced
pressure. The desired product 24 was obtained after tritura-
tion with diethyl ether (3ꢄ5 mL) as white crystals; yield:
150 mg (72%). NMR analyses were identical with those re-
ported in the literature.[38]
HPLC Conditions
HPLC analyses were performed using an Atlantisꢃ T3
column (150ꢄ4.6 mm; 3 mm). Conditions A: mixture of
water+0.1% TFA/MeCN+0.1% TFA in the ratio of 60:40
as eluent; flow 1 mLminÀ1 and diode array detection. Con-
ditions B: mixture of water+0.1% TFA/MeCN+0.1% TFA
gradient mode (ratio 95/05 to 80/20 in 4 min then 80/20 as
eluent); flow 1 mLminÀ1 and UV detection at l=280 nm.
Conditions C: mixture of water+0.1% TFA/MeCN+0.1%
TFA as eluent with a linear gradient in the ratio of 75:25 to
25:75 in 20 min; flow 1 mLminÀ1 and UV detection at l=
360 nm. Chiral HPLC analyses were performed using the
ChiralPakꢃ IC column (250ꢄ4.6 mm) containing the immo-
bilized chiral stationary phase cellulose tris-(3,5-dichloro-
phenylcarbamate). Conditions D: mixture of heptane+
0.1% TFA/i-PrOH+0.1% TFA in the ratio of 80:20 as
eluent; flow 1 mLminÀ1 and diode array detection. Condi-
tions E: mixture of heptane+0.1% TFA/i-PrOH+0.1%
TFA in the ratio of 90:10 as eluent; flow 1 mLminÀ1 and
diode array detection. Conditions F: mixture of heptane+
0.1% TFA/i-PrOH+0.1% TFA in the ratio of 95:05 as
eluent; flow 1 mLminÀ1 and UV detection at l=280 nm.
3-Amino-3-phenylpropanenitrile (as TFA salt) (25): To
a suspension of tert-butyl (2-hydroxy-1-phenylethyl)carba-
mate.[39] (4.64 mmol, 1 equiv., 1.1 g) in dried dichlorome-
thane (9 mL) was added under inert atmosphere at room
temperature triethylamine (6.96 mmol, 1.5 equiv., 968 mL).
A
solution
a
methanesulfonyl chloride (7.52 mmol,
1.6 equiv., 579 mL) in dried dichloromethane (9 mL) was
slowly added to the mixture precooled to 08C. After stirring
for 1 hour at room temperature, the reaction was stopped
by addition of a saturated solution of sodium hydrogen car-
bonate (19 mL). The aqueous phase was extracted with di-
chloromethane (3ꢄ10 mL) and the combined organic layers
were dried over magnesium sulfate, filtered and concentrat-
ed to dryness under reduced pressure. The obtained crude
mesylate (4.64 mmol, 1 equiv.) was dissolved in dimethyl
sulfoxide (26 mL) and potassium cyanide (8.35 mmol,
1.8 equiv., 543 mg) was added. The mixture was stirred for
19 h at 408C then water (25 mL) and diethyl ether (12 mL)
were added. The aqueous phase was extracted with diethyl
ether (4 x10 mL) and the combined organic layers were
washed with brine (15 mL), dried over magnesium sulfate,
filtered and concentrated to dryness under reduced pressure.
Purification of the product by flash chromatography on
silica gel (gradient mode: petroleum ether/ethyl acetate 100/
0 during 5 min then 100/0 to 80/20 in 40 min) afforded the
N-protected 3-amino-3-phenylpropanenitrile as a slightly
yellow solid; yield: 400 mg (34%).
Substrate and Standard Synthesis
3-Hydroxypentanenitrile (21): To a suspension of potassium
cyanide (100 mmol, 5 equiv., 6.50 g) and ammonium chlo-
ride (44 mmol, 2.2 equiv., 2.33 g) in an 8/1 methanol/water
mixture (40 mL) cooled to 08C, was slowly added 2-ethylox-
irane (20 mmol, 1 equiv., 1.74 mL). The mixture was stirred
for 24 h at 708C and then quenched by addition of water
(30 mL) and diethyl ether (30 mL). The aqueous phase was
extracted with diethyl ether (3ꢄ10 mL) and the combined
organic layers were washed with brine (10 mL), dried over
magnesium sulfate, filtered and concentrated to dryness
under reduced pressure. Purification of the product by flash
chromatography on silica gel (gradient mode: petroleum
ether/ethyl acetate 90/10 to 50/50 in 15 min) afforded the de-
sired product 21 as a yellow oil; yield: 1.21 g (60%). NMR
analyses were identical with those reported in the litera-
ture.[36]
3-Hydroxy-3-phenylpropanenitrile (22): The (3-hydroxy-
3-phenylpropanenitrile (22) was synthesized according to
the procedure described by Hua et al.[37]
To a solution of the latter product (0.72 mmol, 1 equiv.,
185 mg) in dried dichloromethane (1.5 mL) were successive-
ly added triethylsilane (3.61 mmol, 5 equiv., 575 mL) and tri-
fluoroacetic acid (9.39 mmol, 13 equiv., 698 mL) under an
inert atmosphere at room temperature. The mixture was
stirred for 15 min and then concentrated to dryness under
reduced pressure. The product (185 mg, quantitative yield)
was obtained as a mixture of desired 3-amino-3-phenylpro-
panenitrile 25 as TFA salt and 3-(tert-butylamino)-3-phenyl-
3-Aminopentanenitrile (as TFA salt) (24): To a suspen-
sion of N-Boc-2-amino-1-butanol (5.3 mmol, 1 equiv., 1.0 g)
in dried dichloromethane (10 mL) under an inert atmos-
phere was added triethylamine (7.9 mmol, 1.5 equiv.,
1.1 mL). The mixture was stirred for 15 min at 08C then a so-
lution a methanesulfonyl chloride (8.6 mmol, 1.6 equiv.,
0.66 mL) in dried dichloromethane (6 mL) was slowly
added. The reaction mixture was stirred for 3 h at room
temperature and then quenched by addition of a saturated
solution of sodium hydrogen carbonate (25 mL). The aque-
1
propanenitrile in the molar ratio 4.7/1. H NMR (MeOD-d4,
300 MHz): d=7.44–7.54 (m, 5H, H-5, H-6, H-7), 4.73 (t, J=
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Carine Vergne-Vaxelaire et al.
FULL PAPERS
7.0 Hz, 1H, H-3), 3.25 (d, J=7.0 Hz, 2H, H-2); 13C NMR
(MeOD-d4, 75 MHz): d=136.0 (C-4), 128.3–131.3 (C-5, C-6,
C-7), 116.8 (C-1), 52.8 (C-3), 24.0 (C-2) (see the Supporting
Information, Figures S3 and S4).
(R)-Methyl 3-cyano-2-phenylpropanoate [(R)-26]: The
(R)-methyl 3-cyano-2-phenylpropanoic acid [(R)-26] was
synthesized from the corresponding (R)-acid[40] and then es-
terified by common reaction with thionyl chloride and meth-
anol; tR [(R)-26]=13.39 min, conditions D.
ylic acid 29a was enantiomerically enriched by resolution
with brucine salts according to the method described by
Curry et al.[44] After two recrystallizations, (R)-3-oxocyclo-
pentanecarboxylic acid was obtained with an enantiomeric
excess of 29% as determined by chiral HPLC (conditions
F).
2-[(cyanomethyl)amino]acetic acid (32): 2-[(Cyanome-
thyl)amino]acetic acid (32) was synthesized according to the
procedure described in the literature.[45a] NMR analyses
were identical with those reported in the literature.[45b]
4-Methoxy-4-oxo-3-phenylbutanoic acid (27): 4-tert-
Butyl 1-methyl 2-phenylsuccinate 33 was synthesized accord-
ing to a similar procedure as described.[41] Methyl 2-phenyla-
cetate (4.50 mmol, 1 equiv., 676 mg) in dried THF (0.65 mL)
was slowly added to a solution of lithium diisopropylamide
generated in situ from n-butyllithium (4.59 mmol,
1.02 equiv., 2.85 mL) and diisopropylamine (4.72 mmol,
1.05 equiv., 667 mL) in 19 mL of dried THF. The solution
was stirred for 45 min at À788C, then tert-butyl bromoace-
tate (13.50 mmol, 3 equiv., 2 mL) was slowly added, the re-
sulting solution was stirred for 20 min at À788C and then al-
lowed to warm up to room temperature over 4 h30 min
under N2. The reaction was quenched with saturated ammo-
nium chloride solution (10 mL) and THF was removed
under reduced pressure. Ethyl acetate (20 mL) was added
and the layers separated. The organic layer was dried
(MgSO4), filtered and concentrated to dryness under re-
duced pressure. Purification of the product by flash chroma-
tography on silica gel (96/4 cyclohexane/EtOAc) afforded 4-
tert-butyl 1-methyl 2-phenylsuccinate 33 as a slightly yellow
oil; yield: 810 mg (68%) (see the Supporting Information,
Determination of Optimum Biocatalytical Reaction
Parameters
Activity of Sphingomonas wittichii nitrilase NIT28
(A5V4Y8) on 26: An aliquot (80–100 mL) of the reaction
mixture was quenched after a specified period of time by
addition of TFA (1% of the total volume), centrifuged to
remove the protein and the supernatant was subjected to
HPLC analysis. The conversion was determined by HPLC
using conditions A [tR (27)=5.64 min, tR (26)=9.16 min].
The ee values of the acid were determined by chiral HPLC
analysis after extraction with ethyl acetate (3ꢄ100 mL for
aliquots of 100 mL), concentration using a vacuum concen-
trator and solubilization of the residue in heptane/i-PrOH
(70 mL; ratio 50:50); conditions D: tR [(R)-27]=6.47 min, tR
[(S)-27]=5.72 min, tR [(S)-26]=11.76 min, tR [(R)-26]=
13.39 min.
Activity of Syntrophobacter fumaroxidans nitrilase
NIT278 (A0LKP2) on 28a: An aliquot (80–100 mL) of the
reaction mixture was quenched after a specified period of
time by addition of TFA (1% of the total volume), centri-
fuged to remove the protein and the supernatant was sub-
jected to HPLC analysis. The conversion was determined by
HPLC using conditions B [tR (28a)=5.48 min, tR (29a)=
5.09 min]. The ee values of the acid were determined by
chiral HPLC analysis after extraction with methyl tert-butyl
ether (5ꢄ100 mL for aliquots of 100 mL), concentration at at-
mospheric pressure until an approximate volume of 70 mL
and injection (20 mL) on chiral HPLC, using conditions F:
tR [(R)-29a]=18.76 min, tR [(S)-29a]=19.96 min.
1
Figures S4 and S5 for H and 13C NMR spectra).
To tert-butyl ester 33 (0.39 mmol, 104 mg) was added
a 4M solution of HCl in dioxane (1.97 mmol, 5 equiv.,
0.5 mL) and the resulting solution was stirred for 48 h at
room temperature. The solution was concentrated under re-
duced pressure. Purification of the product by flash chroma-
tography on silica gel (linear gradient 100/0 to 98/2 CH2Cl2/
MeOH+0.5% HCO2H in 15 min) afforded the desired
product 27 as a white solid; yield: 73 mg (89%).
1
Ester 33: H NMR (CDCl3, 300 MHz): d=7.17–7.27 (m,
5H, H2–H4), 3.95 (dd, J=10.2 and 5.5 Hz, 1H, H-5), 3.60
(s, 3H, H-11), 3.04 (dd, J=16.6 and 10.2 Hz, 1H, H-6a),
2.53 (dd, J=16.6 and 5.5 Hz, 1H, H-6b), 1.33 (s, 9H, H-9);
13C NMR (CDCl3, 75 MHz): d=173.7 (C-10), 170.8 (C-7),
138.0 (C-1), 129.0 (C-2), 128.0 (C-3), 127.7 (C-4), 81.2 (C-8),
52.5 (C-11), 47.5 (C-5), 39.3 (C-6), 28.2 (C-9).
Activity of Sphingomonas wittichii nitrilase NIT158
(A5VE02) on 30: An aliquot (80–100 mL) of the reaction
mixture was quenched after a specified period of time and
derivatized according to the procedure described below. An
aliquot was subjected to HPLC analysis: the conversion was
determined using conditions C: tR (diacid 31)=6.68 min, tR
(serine)=7.70 min, tR (monoacid 32)=9.72 min.
Acid 27: NMR analyses were identical with those report-
ed in the literature.[42]
Racemic 3-oxocyclopentanecarboxylic acid (29a): To
a
suspension of 3-oxocyclopentanecarbonitrile[10a] 28a
Procedure for the Derivatization with 1-Fluoro-2,4-
dinitrobenzene (FDNB)
(1.83 mmol, 1 equiv., 204 mg) in water (1.41 mL) were
added acetic acid (3.41 mL) followed by sulfuric acid
(1.02 mL). The reaction mixture was heated at 1008C for 4 h
until disappearance of the starting material. After addition
of water, the aqueous phase was extracted with ethyl acetate
(3 times) and diethyl ether (9 times). The combined organic
phases were dried over MgSO4, filtered and concentrated
under reduced pressure to afford the acid 29a as a slightly
yellow solid; yield: 220 mg (93%). NMR analyses were iden-
tical with those reported in the literature.[43]
50 mL of the reaction mixture were quenched by addition of
TFA (1 mL). The resulting solution was centrifuged to
remove the proteins. The resulting supernatant (45 mL) was
neutralized with 5M of potassium carbonate solution
(1.5 mL). 1 mL of serine 50 mM was then added as internal
standard followed by EtOH (450 mL), 250 mM of sodium bi-
carbonate solution (50 mL) and a 0.25% solution of FDNB
solution in EtOH (150 mL) (conditions 1), or EtOH
(100 mL), 250 mM of sodium bicarbonate solution (50 mL)
and a 0.25% solution of FDNB solution in EtOH (500 mL)
Enantiomerically enriched 3-oxocyclopentanecarboxylic
acid ACHTUNGTRENNUNG(29a): A portion of racemic 3-oxocyclopentanecarbox-
1776
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Adv. Synth. Catal. 2013, 355, 1763 – 1779

Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools
(conditions 2). The resulting solution was heated at 608C for
3 h with shaking (700 rpm) then quenched with HCl 1M
(25 mL) and filtered off before analysis on reverse-phase
column HPLC.
tion). Purified enzyme was added to bring the concentration
to 0.04 mgmLÀ1 and the reaction (total volume 10 mL) was
shaken (195 rpm, orbital shaking) for 48 h at 378C to ach-
ieve a conversion of 50% as checked by HPLC [conditions
B: tR (acid 29a)=5.09 min, tR (nitrile 28a)=5.48 min]. The
reaction mixture was acidified with TFA (100 mL) and cen-
trifuged to remove the enzyme. The supernatant was basi-
fied to pH 10 by addition of a solution of saturated sodium
bicarbonate and ethyl acetate (15 mL) was added. After de-
cantation, the nitrile was extracted with ethyl acetate (2ꢄ
15 mL) and the combined organic layers were dried
(MgSO4), filtered and concentrated under reduced pressure
to give 16 mg of remaining nitrile (97% of recovery based
on 50% conversion) contaminated with slight amount of
oxidized DTT originating from enzyme preparation. The
basic aqueous layer was acidified to pH 1–2 by addition of
HCl 2M at 48C and then extracted with ethyl acetate (3ꢄ
20 mL). The combined organic layers were dried (MgSO4),
filtered and concentrated under reduced pressure to give,
after flash chromatography on silica gel (99/1 CH2Cl2/
MeOH+0.5% HCO2H), the desired acid 29a as a white
solid; yield: 14 mg (74% based on 50% conversion).
The ee values of acid 29a {48%; tR [(R)-29a]=18.76 min,
tR [(S)-29a]=19.96 min] were determined by chiral HPLC
analysis (conditions F). NMR analyses were identical with
those of the synthetic standard.
Biocatalytic Synthesis (Scale-Up Reactions)
Biocatalytic Synthesis of 4-Methoxy-4-oxo-3-phenyl-
butanoic Acid (27) from Methyl 3-Cyano-2-phenyl-
propanoate (26) by Sphingomonas wittichii Nitrilase
NIT28
In a Greiner tube (50 mL) with a screw cap was poured a so-
lution of 200 mM of methyl 3-cyano-2-phenylpropanoate in
methanol (1.250 mL, 250 mmol, 10 mM final concentration),
an appropriate volume of water and 750 mL of a solution of
potassium phosphate buffer 1M pH 7.3 (30 mM final con-
centration). The purified enzyme was added to bring the
concentration to 0.015 mgmLÀ1 and the reaction (final
volume 25 mL) was shaken (150 rpm, orbital shaking) for
7 h30 min at 308C to achieve a conversion of 50% as
checked by HPLC [conditions A: tR (acid 27)=5.64 min; tR
(nitrile 26)=9.16 min]. The reaction mixture was acidified
with TFA (250 mL) and centrifuged to remove the enzyme.
After addition of ethyl acetate (50 mL) to the supernatant
and decantation, the organic layer was washed with water
(3ꢄ25 mL) to remove salts and then a solution of saturated
sodium bicarbonate (25 mL) was added. After separation of
the two layers, the organic layer was washed with another
10 mL of saturated sodium bicarbonate and kept separately.
The basic aqueous layer was washed with dichloromethane
(3ꢄ10 mL) to remove traces of nitrile, acidified to pH 2 by
addition of HCl 3M at 48C and then extracted with ethyl
acetate (3ꢄ25 mL). The combined organic layers were
washed with brine (10 mL), dried (MgSO4), filtered and con-
centrated under reduced pressure to give the desired acid 27
as a white solid; yield: 27 mg (96% based on 50% conver-
sion).
The kept organic layer was washed with brine (10 mL),
dried (MgSO4), filtered and concentrated under reduced
pressure to give 22 mg of remaining nitrile 26 (93% of re-
covery based on 50% conversion).
The ee values of acid 27 [46%; tR (R)=10.14 min, tR
(S)=8.60 min] and recovered nitrile 26 [42%; tR (S)=
11.76 min, tR (R)=13.39 min] were determined by chiral
HPLC analysis (conditions E and D respectively). NMR
analyses were identical with those of the synthetic standard.
This protocol done on two replicates has been shown to be
reproducible.
This protocol done on two replicates has been shown to
be reproducible.
Biocatalytic Synthesis of 2-[(Cyanomethyl)amino]-
acetic Acid (32) from Iminodiacetonitrile (30) by
Sphingomonas wittichii Nitrilase NIT158
In a Greiner tube (50 mL) with a screw cap was poured a so-
lution of 100 mM of iminodiacetonitrile (30) (3.000 mL,
300 mmol, 30 mM final concentration), an appropriate
volume of water and 300 mL of a solution of potassium phos-
phate buffer 1M pH 7.3 (30 mM final concentration). An
appropriate amount of enzyme as crude cell lysate was
added and the reaction (final volume 10 mL) was shaken
(195 rpm, orbital shaking) for 24 h at 378C. The reaction
mixture was acidified with TFA (100 mL) and centrifuged to
remove the proteins. After concentration of the supernatant,
the residue was purified by flash chromatography on silica
gel (linear gradient 90/10 to 70/30 CH2Cl2/MeOH+0.5%
HCO2H in 15 min) followed by flash chromatography on
C18 (elution H2O) to afford the desired product 32 as
a white solid; yield: 25 mg (73%). NMR analyses were iden-
tical with those of the synthetic standard.
Phylogenetic Analysis
Biocatalytic Synthesis of 3-Oxocyclopentanecarbox-
ylic acid (29a) from 3-Oxocyclopentanenitrile (28a)
by Syntrophobacter fumaroxidans Nitrilase NIT278
Phylogenetic analysis was carried out using a ready-to-use
pipeline (http://www.phylogeny.fr/) chaining programs:
MUSCLE for multiple alignment, PhyML for tree building
and TreeDyn for tree rendering.[46] All the nitrilase Uni-
protKB identifiers are reported in the tree. The nitrilases
from the plant Arabidopsis thaliana (P46010, P32961,
P32962) and from the fungi Aspergillus niger (A9QXE0)
have been used as nitrilase eukaryotic references.[47]
In a Greiner tube (15 mL) with a screw cap was poured a so-
lution of 100 mM of 3-oxocyclopentanenitrile (28a)
(3.000 mL, 300 mmol, 30 mm final concentration), an appro-
priate volume of water and 800 mL of a solution of potassi-
um phosphate buffer 1M pH 7.3 (80 mM final concentra-
Adv. Synth. Catal. 2013, 355, 1763 – 1779
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1777

Carine Vergne-Vaxelaire et al.
FULL PAPERS
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