Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine by Pseudomonas putida

Article abstract of DOI:10.2478/s11696-013-0521-7

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L^-1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L ^-1 h^-1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.

Full text of DOI:10.2478/s11696-013-0521-7

Chemical Papers 68 (6) 739–744 (2014)
DOI: 10.2478/s11696-013-0521-7
ORIGINAL PAPER
Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine
by
a,b
a,b
a
a
a
Xiao-Yan Zhu, Jin-Song Gong, Heng Li, Zhen-Ming Lu, Jin-Song Shi,
a,b
Zheng-Hong Xu*
a
b
School of Pharmaceutical Science, The Key Laboratory of Industrial Biotechnology, Ministry of Education,
Jiangnan University, Wuxi 214122, China
Received 10 September 2013; Revised 25 October 2013; Accepted 29 October 2013
Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production
from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5
◦
◦
◦
◦
and 45 C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30 C, 38 C, and 45 C,
respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min.
The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in
◦
−1
a 1 L system at 30 C and finally, 123 g L of isonicotinic acid were obtained within 200 min without
any by-products. The conversion reaction suffered from the product inhibition effect after the tenth
feeding. The volumetric productivity was 36.9 g L
−
1
−1
h
. P. putida shows significant potential in
nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid
biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production
reported so far.
ꢀ
c 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: biocatalysis, 4-cyanopyridine, isonicotinic acid, nitrilase, Pseudomonas putida
Introduction
through chemical approaches, while the enzymatic hy-
drolysis approach has attracted much attention in re-
cent few years due to its mild reaction conditions,
high specificity, high selectivity and eco-friendliness.
However, only very few organisms including Nocar-
dia globerula NHB-2, Pseudomonas fluorescens C2,
Fusarium solani O1, and Aspergillus niger K10 have
been reported for isonicotinic acid production. On
the other hand, Nocardia globerula NHB-2 nitrilase
has been already used in 4-cyanopyridine biotrans-
formation (Sharma et al., 2012). The authors ap-
plied fed-batch reaction to continuously transform
Among several examples of nitrilase-mediated bio-
catalysis, pathways for the biosynthesis of isonicotinic
acid from 4-cyanopyridine have not been studied ex-
tensively. The effort has been focused on the appli-
cation of this enzyme for the production of its iso-
mer, nicotinic acid, and other carboxylic acids, such
as glycolic acid, acrylic acid, p-hydroxybenzoic acid,
N-acylamino acids, and (R)-(–)-mandelic acid (Ku-
mar & Bhalla, 2013; Chaplin et al., 2004; Gong et al.,
2
012). Thus, the exploration of its practical poten-
◦
tial in isonicotinic acid production is of great signifi-
cance since it plays a functional role in the synthesis
of plant growth regulator inabenfide, antituberculo-
static drug isoniazid, and antidepressant nialamide in
agricultural and pharmaceutical industry (Arai et al.,
4-cyanopyridine at 35 C and 117.9 g (958 mM) of ison-
icotinic acid were finally obtained within 400 min with
an addition of 100 mM of 4-cyanopyridine per 20 min.
The catalyst suffered from substrate inhibition after
the 7th feeding. Very recently, Maksimova and co-
workers isolated a strain of P. fluorescens C2 contain-
ing nitrilase and found that it showed certain specific
2
004; Sharma et al., 2012).
Conventionally, isonicotinic acid was produced
*Corresponding author, e-mail: zhenghxu@jiangnan.edu.cn

7
40
X. Y. Zhu et al./Chemical Papers 68 (6) 739–744 (2014)
activity toward 4-cyanopyridine. About 16 g L−1 of
-cyanopyridine were converted within 300 min by this
strain (Maksimova et al., 2013).
and 1 g of NaCl per liter for 24 h. This preculture
(1 vol. %,) was inoculated to a 250 mL flask con-
taining 55 mL of basal culture medium additionally
4
−
1
In addition, it has been proved that fungal ni-
trilases from the Aspergillus, Fusarium, and Gib-
berella genera have potential in 3-, 4-cyanopyridine
hydrolysis (Gong et al., 2012). These nitrilases show
high specificity towards this sort of nitriles. However,
amide compounds were simultaneously generated in
the conversion reaction. Researchers from the Czech
republic (Vejvoda et al., 2006) prepared isonicotinic
acid using Aspergillus niger K10 nitrilase, which was
highly specific towards 4-cyanopyridine, as the cata-
lyst. The enzyme was found to generate large amounts
of amide from nitrile at an amide/acid molar ratio
of 1/3. For this reason, the authors had to use the
co-immobilization method to remove the by-product;
then, Rhodococcus erythropolis A4 amidase was co-
immobilized on a butyl sepharose column with nitri-
lase. The mole ratio of amide was decreased to approx-
imately 5 %. Another fungal nitrilase from Fusarium
solani O1 afforded < 0.2 mole % of amide when using
the same approach from 2 %. Afterwards, continuous-
stirred membrane reactors were developed using cross
linked F. solani O1 nitrilase and R. erythropolis A4
amidase to further improve the conversion process
supplemented with 1 g L
The incubation was routinely performed at 30 C for
36 h under shaking at 120 min . The harvested cells
were collected by centrifugation (10000g, 10 min) and
washed twice with 100 mM of potassium phosphate
buffer (pH 7.5). The mass of dry cells (DCW) of the
of urea as the inducer.
◦
−
1
◦
resting cells was determined after drying at 115 C for
2 h.
Unless otherwise stated, the standard reaction for
4-cyanopyridine conversion to isonicotinic acid was
performed in 10 mL of 100 mM potassium phos-
◦
phate buffer (pH 7.5) at 30 C with 5 mg of cells and
50 mM of 4-cyanopyridine. The samples were with-
drawn at regular intervals, and the reaction was ter-
minated by adding 10 vol. % of 1 M HCl. Subse-
quently, the samples were centrifuged at 10000g for
5 min. 4-Cyanopyridine and isonicotinic acid in the
supernatant were analyzed by high performance liq-
uid chromatography (HPLC). One unit of nitrilase
activity was defined as the dry cell of 1 mg catalyz-
ing the formation of isonicotinic acid at the rate of
−
1
1
mol min
under the standard assay conditions.
The optical density of culture broth was spectropho-
tometrically determined at 600 nm (Mapada UV-1800,
Shanghai, China).
(Malandra et al., 2009). Interestingly, the purity of
isonicotinic acid was further increased to 99.9 %. How-
ever, the by-product, isonicotinamide, still could not
be fully removed while the cost increased and the pro-
cedure became complicated.
In this work, Pseudomonas putida was used as
the biocatalyst for the biotransformation of 4-cyano-
pyridine to isonicotinic acid (Zhu et al., 2013a). This
strain was previously isolated from soil in our labo-
ratory. It showed significant catalytic efficiency on 3-
and 4-cyanopyridine hydrolysis, while no amide was
formed in the reaction mixture. Its nitrilase gene was
identified by our group and the conversion reaction
was proved to be mediated by nitrilase (Zhu et al.,
The effects of pH, temperature, substrate concen-
tration, and resting cell concentration on the conver-
sion reaction were studied. The pH range of the reac-
tion was from 5.0 to 9.0 with sodium acetate buffer
(pH 5.0–6.0), potassium phosphate buffer (pH 6.0–
7.5), or Tris–HCl buffer (pH 7.5–9.0). The optimum
reaction temperature was determined by carrying out
◦
the reaction at various temperatures, from 20 C to
◦
◦
◦
60 C. The cells were pre-incubated at 30 C, 38 C,
and 45 C to determine the thermostability of the en-
◦
zyme. The cell suspension was sampled at regular in-
tervals to measure the residual activity which was
determined by comparison with the initial nitrilase
activity of unincubated cells. The reaction systems,
including 4-cyanopyridine in different concentrations
2
013b). Reaction conditions of the 4-cyanopyridine
conversion were investigated and a bench-scale pro-
cess was carried out applying the fed-batch method.
(50 mM to 300 mM), were used to evaluate the ef-
Experimental
fect of substrate concentration. To obtain the opti-
mal cell concentration for substrate conversion, vari-
ous amounts of resting cells (1 mg of DCW per mL to
3 mg of DCW per mL) were tested for the complete
conversion of 100 mM of 4-cyanopyridine to isonico-
tinic acid.
4
-Cyanopyridine, isonicotinic acid, and isonicoti-
namide were purchased from Sigma–Aldrich Chemical
Co. (St. Louis, MO, USA). All other analytical-grade
reagents and chemicals were obtained from commer-
cial sources and they were used in the present study
without further purification.
In all investigations, P. putida CGMCC3830 was
employed. This strain was deposited in the China Gen-
eral Microbiological Culture Collection Center (Bei-
The bioconversion reaction was performed us-
ing the resting cell concentration of 3.0 mg of
◦
DCW per mL at 30 C under constant stirring.
4-Cyanopyridine was added into the 1 L reaction mix-
ture in the fed-batch mode and the conversion reac-
tion was monitored by HPLC. It was carried out in
a 3 L fermentor (Baoxing Bio-Engineering Equipment
Co., Shanghai, China). The agitation speed of the fer-
◦
jing, China). It was precultivated at 30 C in a basal
medium (pH 6.0) containing 12.5 g of glycerol, 12.5 g
of tryptone, 5 g of yeast extract, 1 g of KH2PO4,

X. Y. Zhu et al./Chemical Papers 68 (6) 739–744 (2014)
741
Fig. 1. Effect of pH (A) on nitrilase acitivity of P. putida: relative activity was expressed as the percentage of the activity at pH
−
1
7
(
.5 (specific activity of 5.2 U mg ). Symbols: sodium acetate buffer ( ), potassium phosphate buffer ( ), Tris–HCl buffer
•
ꢀ). Effect of temperature (B) on nitrilase acitivity of P. putida: relative activity was expressed as the percentage of the
◦
−1
activity at 45 C (specific activity 8.2 U mg ).
mentor was maintained at 400 min−1, at a constant
◦
temperature of 30 C.
The amounts of 4-cyanopyridine and isonicotinic
acid in the reaction mixture were determined by
HPLC (UltiMate 3000 liquid chromatography system;
Dionex, Sunnyvale, CA, USA). HPLC analysis was
performed employing a Chromeleon chromatography
data system, using an Atlantis dC18 column (particle
size of 5.0 m; dimensions of 150 mm × 4.6 mm; Wa-
ters, Milford, MA, USA) at the wavelength of 268 nm.
The mobile phase was acetonitrile: 0.025 vol. % phos-
−
1
phoric acid (ϕr = 6/4) at the flow rate of 0.5 mL min
at 30 C.
◦
Fig. 2. Thermostability of intracellular nitrilase of P. putida.
Variation of nitrilase relative activity with time at dif-
Results
◦
◦
◦
), and 45 C
ferent temperatures: 30 C ( ), 38 C (
ꢀ).
•
(
Effect of reaction conditions on bioconversion
P. putida nitrilase is able to hydrolyze 4-cyano-
pyridine in a relative wide pH range and it shows the
maximal activity at pH 7.5 with the potassium phos-
phate or Tris–HCl buffer, while almost identical ac-
tivities were observed using these two buffer solutions
nant nitrilase, where the reaction temperature and pH
of purified recombinant nitrilase with 3-cyanopyridine
as substrate were 40 C and pH 7.5, respectively (Zhu
◦
et al., 2013b).
(
6
Fig. 1A). The activity decreased sharply at pH below
.0, and only 14 % of the activity was determined at
In order to investigate the thermostability of
P. putida nitrilase, resting cells were preincubated at
◦
◦
◦
pH 5.5. Interestingly, this nitrilase preferred alkaline
conditions and 43 % of activity was retained at pH
30 C, 38 C, and 45 C, respectively. Fig. 2 shows that
◦
the intracellular enzyme was stable at 30 C and re-
9
.0. No spontaneous degradation of 4-cyanopyridine
tained 93 % of its activity after a 12 h incubation.
was detected under acidic or alkalic conditions.
P. putida nitrilase showed higher catalytic activ-
ity for 4-cyanopyridine conversion in the temperature
The half-life (t1/2) of this intracellular nitrilase was
◦
◦
calculated to be 93.3 h (30 C), 33.9 h (38 C), and
◦
9.5 h (45 C), according to the residual activity plot-
◦
range of 40–50 C (Fig. 1B); the maximum activity was
ted against time, while the recombinant nitrilase of
this strain showed poorer stability than the wild one
(Zhu et al., 2013b). Taking into account the enzyme
◦
achieved at 45 C. The nitrilase activity of P. putida
◦
◦
increased from 20 C to 45 C with the reaction tem-
perature, while the activity decreased rapidly when
the temperature exceeded 45 C. It can be attributed
◦
activity and thermostability, the temperature of 30 C
◦
was chosen as the reaction temperature in further ex-
periments.
To obtain the optimal substrate concentration,
the reaction was carried out at different concentra-
to the inactivation of intracellular nitrilase at higher
temperatures. Furthermore, there is a certain differ-
ence in the optimum conditions for wild and recombi-

7
42
X. Y. Zhu et al./Chemical Papers 68 (6) 739–744 (2014)
Fig. 3. Effect of 4-cyanopyridine concentration (A) on its bioconversion. Initial 4-cyanopyridine concentration: 50 mM (
mM ( ), 200 mM (ꢀ), and 300 mM ( ). Effect of resting cells concentration (B) on 4-cyanopyridine bioconversion. Resting
cell concentration in mg of DCW per mL: 1 ( ), 2 (
), and 3 (ꢀ).
•
), 100
•
tions of 4-cyanopyridine ranging from 50 mM to
00 mM (Fig. 3A). Complete conversion of 100 mM
3
of 4-cyanopyridine was achieved within 1 h. The
rate of isonicotinic acid synthesis decreased when
4
-cyanopyridine concentration was higher than 100 mM.
This could be caused by the toxicity and inhibition
due to high 4-cyanopyridine concentration. Finally,
1
00 mM of 4-cyanopyridine were chosen as the op-
timal substrate concentration.
Fig. 3B shows that an increased production of ison-
icotinic acid was observed with the cell concentration
increase from 1 mg of DCW per mL to 3 mg of DCW
per mL. A 66 % conversion was observed for 100 mM
of 4-cyanopyridine within 20 min when using 2 mg of
DCW per mL; while 3 mg of DCW per mL afforded a
Fig. 4. Fed-batch reaction profile of 4-cyanopyridine biocon-
version by P. putida nitrilase. Concentration of: isoni-
cotinic acid ( ), 4-cyanopyridine ( ).
•
1
00 % 4-cyanopyridine conversion. Therefore, the con-
centration of 3 mg of DCW per mL was chosen as the
optimal cell concentration in further experiments.
of 100 mM of 4-cyanopyridine to the reaction mix-
ture, the conversion process efficiency decreased due
to product inhibition and enzyme inactivation. The
Fed-batch reaction for isonicotinic acid pro-
duction
1
00 % conversion of the newly added 4-cyanopyridine
−
1
Complete conversion of 100 mM of 4-cyanopyridine
into isonicotinic acid was achieved within 20 min when
using P. putida resting cells as the catalyst. The fed-
was observed after 60 min. Finally, 172 g L of ison-
icotinic acid (1400 mM) were accumulated within 380
min by 14 substrate feedings (Fig. 4). No isonicoti-
namide was detected in the reaction mixture.
◦
batch reaction was thus carried out at 30 C in 1 L
of potassium phosphate buffer solution (100 mM, pH
7
.5). The feeding rate of 4-cyanopyridine in the fed-
Discussion
batch reaction was 100 mM per 20 min. The product
inhibition (isonicotinic acid) effect began to occur af-
In recent years, nitrilase-mediated biocatalysis us-
ing whole microbial cells or purified enzymes as
the catalysts has been paid substantial attention by
many academic institutions and industrial companies
(Martínková & Křen, 2010). However, their appli-
cations were hindered by several limitations includ-
ing relatively low catalytic activity, poor operational
stability, by-products generation, etc. (Gong et al.,
2012). A strain of P. putida CGMCC3830 harbor-
−
1
ter the tenth feeding. The concentration of 123 g L
1000 mM) of isonicotinic acid was obtained from
the corresponding 4-cyanopyridine within 200 min
(
(
Fig. 4). The volumetric productivity was calculated
−
1
−1
to be 36.9 g L
h .
However, the addition of substrate was still car-
ried out in order to fully explore the potential of this
catalyst. After the thirteenth and fourteenth feeding

X. Y. Zhu et al./Chemical Papers 68 (6) 739–744 (2014)
743
ing outstanding nitrilase activity while no amide was
by-produced has been isolated, and the application
studies showed its significant potential in enzymatic
conversion of 3- and 4-cyanopyridine into the corre-
sponding acids (Zhu et al., 2013a). The current re-
search work is aimed at the exploration of the poten-
tial of P. putida for isonicotinic acid synthesis from 4-
cyanopyridine. Although there are a number of studies
on Pseudomonas nitrilase available (Layh et al., 1998;
Kiziak et al., 2005; Banerjee et al., 2006), it is the
first report on employing P. putida as a biocatalyst for
isonicotinic acid biosynthesis. This green process is an
attractive alternative to the chemical process owing to
the mild reaction conditions and high conversion rate.
In addition, this strain has been proved as promising
for its catalytic efficiency.
version suffered from substrate inhibition in the fed-
batch reaction (200 mM of substrate feed per 40 min)
and ultimately resulted in the formation of 729 mM
−
1
of isonicotinic acid (89.8 g L ) (Sharma et al., 2012).
Furthermore, a subsequent substrate feeding was per-
−
1
formed in this study, and 172 g L of isonicotinic acid
were obtained within 380 min. The high isonicotinic
acid production can be derived from the good product
tolerance as well as its moderate thermostability.
Conclusions
The present study introduces a new biocatalyst
that can be used for the production of isonicotinic
acid through bioconversion of 4-cyanopyridine. This
is the first report about isonicotinic acid biosynthesis
by P. putida. Moreover, the aforementioned proper-
ties indicate that P. putida CGMCC3830 is a promis-
ing biocatalyst for enzymatic production of isonico-
tinic acid. P. putida nitrilase displayed outstanding
catalytic activity towards 4-cyanopyridine. Also, this
catalyst supported high isonicotinic acid production
due to the attractive feeding batches, which can be
derived from the product tolerance as well as its ther-
mostability. Subsequent gene expression and protein
engineering strategy studies are undergoing to further
improve the stability of this nitrilase and thus enhance
its catalytic efficiency towards nitriles.
Product concentration is one of the limiting factors
of biotransformation process (Gong et al., 2012). In
the present study, this nitrilase showed good product
tolerance. The reaction suffered from product inhibi-
tion after the tenth feeding (1000 mM). With regard
to N. globerula NHB-2, the rate of 4-cyanopyridine hy-
drolyzation decreased to 60 % when 700 mM of isoni-
cotinic acid were accumulated (Sharma et al., 2012).
Moreover, in the conversion process of P. putida,
the formation of the by-product isonicotinamide was
not detected. This was superior to nitrilases of A.
niger K10 and F. solani O1, which generated 25 %
and 2 % of the by-product isonicotinamide in the total
products, respectively (Vejvoda et al., 2006). Here, the
downstream process for by-product removal could be
avoided and the purity of the acid product improved.
The resting cells harboring nitrilase showed a mod-
Acknowledgements. This work was financially supported
by the National Natural Science Foundation of China (No.
21206055), the National High Technology Research and Devel-
opment Program of China (No. 2012AA022204C), the Fun-
damental Research Funds for the Central Universities (No.
JUSRP111A51), and the Research and Innovation Project for
College Graduates of the Jiangsu Province (No. CXLX12 0731).
◦
erate thermostability at 30 C, which enabled the oper-
ation of the fed-batch process for continuous produc-
tion of isonicotinic acid in practical applications. The
References
−
1
concentration of 123 g L of isonicotinic acid from the
corresponding 4-cyanopyridine was achieved through
ten substrate feedings within 200 min. The volumet-
Arai, M., Alavi, Y. I. H., Mendoza, J., Billker, O., & Sin-
den, R. E. (2004). Isonicotinic acid hydrazide: an anti-
tuberculosis drug inhibits malarial transmission in the
mosquito gut. Experimental Parasitology, 106, 30–36. DOI:
−
1
−1
. Maksimova et al.
ric productivity was 36.9 g L
2013) isolated a P. fluorescens C2 harboring nitrilase
which was used for 4-cyanopyridine hydrolysis. The re-
h
(
10.1016/j.exppara.2004.01.002.
−
1
Banerjee, A., Kaul, P., & Banerjee, U. C. (2006). Enhancing the
catalytic potential of nitrilase from Pseudomonas putida for
stereoselective nitrile hydrolysis. Applied Microbiology and
Biotechnology, 72, 77–87. DOI: 10.1007/s00253-005-0255-8.
Chaplin, J. A., Levin, M. D., Morgan, B., Farid, N., Li,
J., Zhu, Z., McQuaid, J., Nicholson, L. W., Rand, C.
A., & Burk, M. J. (2004). Chemoenzymatic approaches
to the dynamic kinetic asymmetric synthesis of aromatic
amino acids. Tetrahedron: Asymmetry, 15, 2793–2796. DOI:
sults showed that about 16 g L of 4-cyanopyridine
were converted within 300 min; however, there are no
further application studies on isonicotinic acid produc-
tion by this strain. On the other hand, Sharma et al.
(2012) observed substrate inhibition after the seventh
feeding using N. globerula NHB-2 and finally obtained
−
1
a total of 958 mM of isonicotinic acid (117.9 g L
)
within 400 min. Their fed-batch reaction was scaled
up to 1 L and complete conversion of 700 mM of 4-
cyanopyridine to the corresponding isonicotinic acid
10.1016/j.tetasy.2004.07.060.
Gong, J. S., Lu, Z. M., Li, H., Shi, J. S., Zhou, Z. M., & Xu,
Z. H. (2012). Nitrilases in nitrile biocatalysis: recent progress
and forthcoming research. Microbial Cell Factories, 11, 142.
DOI: 10.1186/1475-2859-11-142.
−
1
(
86.2 g L ) was obtained in 140 min after seven feed-
ings. On the other hand, Sharma and co-workers si-
multaneously attempted the conversion of 200 mM of
Kiziak, C., Conradt, D., Stolz, A., Mattes, R., & Klein, J.
(2005). Nitrilase from Pseudomonas fluorescens EBC191:
4
-cyanopyridine using resting cell in the reaction; they
cloning and heterologous expression of the gene and biochem-
ical characterization of the recombinant enzyme. Microbiol-
ogy, 151, 3639–3648. DOI: 10.1099/mic.0.28246-0.
found that complete conversion into isonicotinic acid
−
1
(
24.6 g L ) was achieved within 40 min. The con-

7
44
X. Y. Zhu et al./Chemical Papers 68 (6) 739–744 (2014)
Kumar, V., & Bhalla, T. C. (2013). Transformation of p-
hydroxybenzonitrile to p-hydroxybenzoic acid using nitrilase
activity of Gordonia terrae. Biocatalysis and Biotransforma-
tion, 31, 42–48. DOI: 10.3109/10242422.2012.757761.
Sharma, N., Sharma, M., & Bhalla, T. (2012). Nocardia
globerula NHB-2 nitrilase catalysed biotransformation of 4-
cyanopyridine to isonicotinic acid. AMB Express, 2, 25. DOI:
10.1186/2191-0855-2-25.
Layh, N., Parratt, J., & Willetts, A. (1998). Characteriza-
tion and partial purification of an enantioselective arylace-
tonitrilase from Pseudomonas fluorescens DSM 7155. Jour-
nal of Molecular Catalysis B: Enzymatic, 5, 467–474. DOI:
Vejvoda, V., Kaplan, O., Kubáč, D., Křen, V., & Martínková,
L. (2006). Immobilization of fungal nitrilase and bacterial
amidase – two enzymes working in accord. Biocatalysis and
Biotransformation, 24, 414–418. DOI: 10.1080/102424206010
33910.
10.1016/s1381-1177(98)00075-7.
Maksimova, Yu. G., Vasilyev, D. M., Ovechkina, G. V., Mak-
simov, A. Yu., & Demakov, V. A. (2013). Transformation
of 2- and 4-cyanopyridines by free and immobilized cells of
nitrile-hydrolyzing bacteria. Applied Biochemistry and Mi-
crobiology, 49, 347–351. DOI: 10.1134/s000368381304008x.
Malandra, A., Cantarella, M., Kaplan, O., Vejvoda, V., Uh-
náková, B., Štěpánková, B., Kubáč, D., & Martínková, L.
Zhu, X. Y., Gong, J. S., Li, H., Lu, Z. M., Zhou, Z. M., Shi,
J. S., & Xu, Z. H. (2013a). Screening, identification and
culture optimization of a newly isolated aromatic nitrilase-
producing bacterium – Pseudomonas putida CGMCC3830.
Chinese Journal of Biotechnology, in press.
Zhu, X. Y., Gong, J. S., Li, H., Lu, Z. M., Zhou, Z. M.,
Shi, J. S., & Xu, Z. H. (2013b). Characterization and func-
tional cloning of an aromatic nitrilase from Pseudomonas
putida CGMCC3830 with high conversion efficiency toward
cyanopyridine. Journal of Molecular Catalysis B: Enzymatic,
97, 175–183. DOI: 10.1016/j.molcatb.2013.08.012.
(2009). Continuous hydrolysis of 4-cyanopyridine by nitri-
lases from Fusarium solani O1 and Aspergillus niger K10.
Applied Microbiology and Biotechnology, 85, 277–284. DOI:
10.1007/s00253-009-2073-x.
Martínková, L., & Křen, V. (2010). Biotransformations with
nitrilases. Current Opinion in Chemical Biology, 14, 130–
137. DOI: 10.1016/j.cbpa.2009.11.018.