Enhanced catalytic efficiency of nitrilase from Acidovorax facilis 72W and application in bioconversion of 3-cyanopyridine to nicotinic acid

Article abstract of DOI:10.1016/j.molcatb.2017.03.010

In this study, the catalytic efficiency using NitA from Acidovorax facilis 72W for nicotinic acid (NA) production was investigated and further improved by site-directed mutagenesis. Results showed that the specific activity of mutated NitA-C2 (F168V-S192F) towards 3-cyanopyridine increased 5-fold to 35 U mg^?1 protein. Further characterization of the biochemical properties of both nitrilases showed the optimal pH and temperature were 6.0–8.0 and 60 °C, respectively, whereas the pH and thermal stability of NitA-C2 were decreased. Finally, whole cell catalysis was adopted for NA production and a 100% conversion yield was achieved under 0.1 mol L^?1 3-cyanopyridine for both strains. Besides, the conversion rate by E. coli BL21 (DE3-pET-nitA-C2) reached to 1.0 mmol min^?1 g^?1 wet cell weight, which was 3-fold higher than that by E. coli BL21 (DE3-pET-nitA). These results indicated that the mutated NitA-C2 was a promising candidate which holds potential application in biological NA production.

Full text of DOI:10.1016/j.molcatb.2017.03.010

Accepted Manuscript
Title: Enhanced catalytic efficiency of nitrilase from
Acidovorax facilis 72W and application in bioconversion of
3-cyanopyridine to nicotinic acid
Authors: He Li, Weiliang Dong, Yue Zhang, Kuan Liu,
Wenming Zhang, Min Zhang, Jiangfeng Ma, Min Jiang
PII:
S1381-1177(17)30042-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2017.03.010
MOLCAB 3541
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
10-11-2016
6-3-2017
24-3-2017
Please cite this article as: He Li, Weiliang Dong, Yue Zhang, Kuan Liu,
Wenming Zhang, Min Zhang, Jiangfeng Ma, Min Jiang, Enhanced catalytic
efficiency of nitrilase from Acidovorax facilis 72W and application in bioconversion
of 3-cyanopyridine to nicotinic acid, Journal of Molecular Catalysis B:
Enzymatichttp://dx.doi.org/10.1016/j.molcatb.2017.03.010
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Enhanced catalytic efficiency of nitrilase from Acidovorax facilis
72W and application in bioconversion of 3-cyanopyridine to
nicotinic acid
a
a,b
a
a
a,b
†
†
He Li , Weiliang Dong , Yue Zhang , Kuan Liu , Wenming Zhang , Min
Zhanga,b, Jiangfeng Maa,b*, Min Jianga,b*
aState Key Laboratory of Materials-Oriented Chemical Engineering, College of
Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing,
211816, P.R. China
bJiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),
Nanjing Tech University , Nanjing, 211816,P.R. China
†These authors have contributed equally to this work.
*Corresponding authors at: State Key Laboratory of Materials-Oriented Chemical
Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Puzhu South Road 30#, Nanjing 211816, P. R. China.
E-mail addresses: bioengine@njtech.edu.cn (M. Jiang), majiangfeng@njtech.edu.cn (J. Ma).
1

Graphical abstract
 Highlights


The novel nitrilase gene nit was cloned from Acidovorax facilis 72W.
Site-directed mutagenesis was adopted to enhance the specific activity.
2



The recombinant enzyme NitA and NitA-C2 was expressed, purified and
characterized.
NitA-C2 had better activity for applications in biological production of NA.
Abstract
In this study, the catalytic efficiency using NitA from Acidovorax facilis 72W for
nicotinic acid (NA) production was investigated and further improved by site-
directed mutagenesis. Results showed that the specific activity of mutated NitA-C2
(F168V-S192F) towards 3-cyanopyridine increased 5-fold to 35 U mg-1 protein.
Further characterization of the biochemical properties of both nitrilases showed the
optimal pH and temperature were 6.0-8.0 and 60℃, respectively, whereas the pH
and thermal stability of NitA-C2 were decreased. Finally, whole cell catalysis was
adopted for NA production and a 100% conversion yield was achieved under 0.1
mol L-1 3-cyanopyridine for both strains. Besides, the conversion rate by E. coli
BL21 (DE3-pET-nitA-C2) reached to 1.0 mmol min-1 g-1 wet cell weight, which
was 3-fold higher than that by E. coli BL21 (DE3-pET-nitA). These results
indicated that the mutated NitA-C2 was a promising candidate which holds
potential application in biological NA production.
Keywords: Nitrilase; Acidovorax facilis 72W; Site-directed mutagenesis; Nicotinic
acid
3

1. Introduction
Nicotinic acid (NA, 3-pyridine carboxylic acid) is an essential nutrient existing in the
human body and animal which has wide applications in pharmaceuticals, food and
forages industries [1]. The annual world demand of nicotinic acid and its derivatives
is estimated to be 22,000 tons [2]. Traditionally, NA is synthesized by chemical
process which involved either liquid phase oxidation of 2-methyl-5-ethyl pyridine
(MEP) or gas-phase oxidation of picoline to nicotinic acid with nitric acid [3,4].
However, extreme environmental factors (high temperature and high pressure) were
required and by-products were formed during the chemical synthesis process which
not only increased the whole cost but also added difficulty in the downstream
purification stage [3,4]. Thus more efficient and environmental-friendly methods were
needed.
During the past decades, several biocatalytic processes with the advantages of
cleaner, safer and more selective were developed for NA production [5-9]. Among
which, utilization of nitrilase for NA production from 3-cyanopyridine was given
much attention since the whole process doesn’t require an extended reaction period or
multiple reaction steps. Indeed, utilization of nitrilase for fine organic chemicals
production has became a hot research spot and it is regarded the most promising
alternative to chemical hydrolysis [10,11]. Since the first report of using the nitrilase-
mediated bioprocess for NA synthesis by resting Rhodococcus rhodochrous Jl [12],
4

various nitrilases from different microorganisms were discovered afterwards, mainly
including Nocardia globerula NHB-2 [13], Fusarium proliferatum ZJB-09150 [9],
Rhodococcus sp. NDB 1165 [14] and Alcaligenes faecalis MTCC 126 [15]. Besides,
technologies such as optimization of reaction condition, cell immobilization [15] and
utilization of continuously stirred tank UF-membrane bioreactors (CSMRs) [6] were
applied to increase the whole process competitiveness. However, although much
progress has been made during the past decades, poor substrate/product tolerance, low
specific activity and poor stability remained as the main hurdles for its practical
application and searching for nitrilases with high catalytic efficiency is always in
demand.
NitA, a nitrilase from gram-negative Acidovorax facilis 72W (ATCC 55746),
is a classical nitrilase and it exhibited high catalytic efficiency towards a number of
substrates [16]. For example, when applied in 4-cyanopentanoic acid production, a
high volumetric productivity of 79 g L-1 h-1 was achieved by using immobilized
recombinant Escherichia coli SW91 which expressed NitA [17]. As to glycolate
bioconversion, the specific activity reached to 490 U g-1 cells and further increased
by 15-fold after protein engineering [18]. On the other hand, NitA had good
thermal stability and substrate/product tolerance [19] which endowed it to be a
potential candidate fitting for industrial production. However, this excellent NitA
from A. facilis 72W had not been applied to the hydrolysis of 3-cyanopyridine to
5

NA which inspired us to explore the possibility of using NitA for enzymatic NA
production.
In the present study, the feasibility of using NitA from A. facilis72W for NA
production was investigated, and its catalytic properties were characterized after
purification of the nitrilase by Ni2+-NTA resin. Furthermore, site-directed
mutagenesis was adopted to enhance the specific activity for 3-cyanopyridine, and
the conversion rates of 3-cyanopyridine into NA were evaluated and compared with
other researches.
2. Materials and methods
2.1 Chemicals and media.
Peptone and yeast extract were purchased from Oxoid Co. Ltd. (Beijing,
China). The substrates including 3-cyanopyridine and nicotinic acid were
purchased from TCI Co. Ltd. (Shanghai, China). Other chemical reagents were
purchased from Lingfeng Chemical Reagents Co. Ltd. (Shanghai, China). All
molecular reagents were purchased from TaKaRa Co. Ltd. (Dalian, China) and all
chemicals used in this study were of analytical grade or a higher purity.
The components of Luria-Bertani (LB) medium were as follows: 10 g L-1
Tryptone, 5 g L-1 Yeast and 5 g L-1 NaCl. LB medium was used to culture E. coli
strains throughout the experiments.
2.2 Strain, plasmid and primers.
6

The gene sequence (GenBank accession No. DQ444267) of NitA from A.
facilis 72W was obtained from National Center for Biotechnology Information
(NCBI) and synthesized by Genscript Co. Ltd. (Nanjing, China). The plasmid
pET28a (+) was used for expression with a His6-tag at both of C- and N-terminal.
E. coli BL21 (DE3) was used as the host strain and all the primers used in the study
were listed in Table 1.
2.3 Amino acid sequences and structure analysis.
Basic Local Alignment Search Tool (BLAST, www.ncbi.nlm.nih.gov/blast)
and DNAMAN version 5.2 (Lynnon Biosoft, Quebec, Canada) were employed for
the GenBank search, identity assessment, and protein domain determination. The
three-dimensional homology model of nitrilase was constructed using the software
of Molecular Operating Environment (MOE) based on the crystal structure of a
nitrilase from Syechocystis sp. PCC6803 (PDB accession No. 3WUY). The protein
structure was minimized using MMFF94X Forcefield. The 3D Structure of the
substrate, 3-cyanopyridine, was downloaded from the PubChem database after
optimization by adding hydrogens and removing water molecules [20].
2.4 Construction of expression plasmids and site-directed mutagenesis.
The nitA gene was amplified using the primers P1/P2 listed in Table 1. The
PCR reaction was carried out at 94℃for 10 min, followed by 35 cycles of 94℃for
1 min, 55℃for 1 min, 72℃for 1 min; and then 72℃for 10 min as the final
7

elongation step in a PCR Amplifier (TGrandient, Biometra). The PCR products
were separated and purified with the DNA Gel Extraction Kit (Axygen, America)
and the purified PCR products as well as the vector pET-28a (+) were digested with
Nde I and Sal I. Then the digested products were ligated to construct the pET-nitA
plasmid by T4-DNA ligase and the new plasmid was transformed into the
competent E. coli BL21 (DE3) to obtain the recombinant designated as E. coli
BL21 (DE3-pET-nitA).
Site-directed mutagenesis was carried out by overlap extension PCR [21]. The
forward primer F168V-F and the reverse primer F168V-R were a pair of
overlapping complementary primers which were used to obtain the mutant with the
168th amino acid mutated from phenylalanine to valine. Then, serine in 192th site
was replaced by Phe using primers S192F-F and S192F-R to achieve the final
mutant designed as NitA-C2. The mutated fragment was inserted into plasmid pET-
28a (+) and transformed into the competent E. coli BL21(DE3) as described above.
The recombinant strain was designated as E. coli BL21 (DE3-pET-nitA-C2).
2.5 Expression, purification and assay of enzyme activity.
The seed cultures of E. coli BL21 (DE3-pET-nitA) and BL21 (DE3-pET-nitA-
C2) were initiated by incubating in 5 mL LB medium supplemented with 30 μg mL-1
kanamycin. After aerobic culturing at 37℃for 12 h, a 1% inoculum was transferred
into a 500 mL Erlenmeyer flask containing 100 mL LB medium supplemented with
8

30 μg mL-1 kanamycin. 0.1 mmol L-1 isopropyl-β-D-thiogalactopyranoside (IPTG)
was added to induce the nitrilase expression when OD600 reached between 0.4 and 0.8.
After induction at 18℃and 200 rpm for 20 h, cells were harvested by centrifugation
(Eppendorf Centrifuge 5810 R, F-34-6-38 rotor, Brinkman Instruments Inc. USA) at
8085 ×g and 4℃for 10 min and washed twice with 100 mmol L-1 PBS (pH 7.0). Then
cells were disrupted by a scientz-II D ultrasonic generator (Ningbo Scientz Biotech.
Co. Ltd., Ningbo, China) in the ice water mixture for 10 min at a power output of 300
W, and cell debris was removed by centrifugation at 8085 ×g and 4℃for 20 min.
The recombinant NitA and NitA-C2 with His6-tag were purified by Ni2+-NTA
resin (Qiagen, Valencia, CA, USA) using Biomolecular Liquid Chromatography
System (AKTA design, GE Healthcare, US). Non-target proteins were removed by
buffer NPI-20 (50 mmol L-1 NaH2PO4, 300 mmol L-1 NaCl, 20 mmol L-1 imidazole,
pH=8.0). Afterwards, the target protein with His6-tag was eluted with buffer NPI-
250 (50 mmol L-1 NaH2PO4, 300 mmol L-1 NaCl, 250 mmol L-1 imidazole,
pH=8.0). These purified proteins were collected and dialyzed against 100 mmol L-1
PBS buffer (pH 7.0) overnight at 4℃to remove imidazole with dialysis membrane
of MwCO3500.
Protein concentrations were determined using the microspectrophotometer
K5500 (Kaiao Tech. Co. Ltd., Beijing, China). The proteins were electrophoresed
by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and
visualized by staining with Coomassie Brilliant Blue R-250. SDS-PAGE was
9

performed on a 12% gel using electrophoresis apparatus at 80 V for the first 60 min
and followed by 120 V for the next 120 min.
The standard enzyme assays were carried out by mixing 20 μg purified
enzyme in 1.0 mL of 100 mmol L-1 PBS (pH 7.0) containing 100 mmol L-1 3-
cyanopyridine. The reaction mixture was incubated at 40℃for 10 min and the
reaction was terminated by adding 2 mol L-1 HCl. The activity was determined by
+
detecting NH4 concentration in 630 nm using phenol-hypochlorite reaction [22].
One unit of the enzyme activity towards 3-cyanopyridine is defined as the amount
of enzyme required to produce 1 μmol of NA per minute.
2.6 Biochemical properties of the purified NitA and NitA-C2.
2.6.1 Substrate specificity.
The substrate specificity of NitA and NitA-C2 were determined using 3-
cyanopyridine, iminodiacetonitrile, fumarodinitrile, 2-methylglutaronitrile,
succinonitrile, 4-chlorobenzonitrile, mandelonitrile and 3-phenylethylcyanide. The
activity for 3-cyanopyridine at different concentrations (5-40 mmol L-1) was
determined as described above. The kinetic constants Km and kcat were calculated
using a Lineweaver-Burk plot [17]. The reaction was performed by the standard
enzyme assays as described above.
2.6.2 Effects of temperature and pH on activity of NitA and NitA-C2.
10

The optimal reaction pH was assessed at 40℃with pH values varying from 4.0 to
10.0 by using several buffers, including 50 mmol L-1 citrate buffer (pH 4.0-6.0), 100
mmol L-1 PBS (pH 6.0-8.0), and 50 mmol L-1 glycine-NaOH buffer (pH 8.0-10.0).
The effects of the temperature on NitA and NitA-C2 activity were determined at the
optimal pH with temperatures varying from 30℃to 80℃. To measure the pH stability,
the enzyme was incubated at 4℃for 24 h in different buffers and the residual activity
was determined using the standard assay as described above. The thermal stability
assays were assessed by incubating the enzyme preparations at different temperatures
for a certain time. Non-heated enzyme was used as the control (100%).
2.6.3 Effects of metal ions and chemicals agents on catalytic activity of NitA and
NitA-C2.
Purified NitA and NitA-C2 were treated with 1 mmol L-1 EDTA for 5 h at 4℃
and then dialyzed against 100 mmol L-1 PBS (pH 7.0) to remove EDTA. The
activities were assayed as described above and compared to the activity of untreated
enzyme solution incubated under the same conditions. For reactivation, the metal-free
enzyme was incubated with divalent metal ions (Fe2+, Ba2+, Cu2+, Co2+, Ni2+, Zn2+,
Ca2+, Mg2+ and Mn2+) at a final concentration of 1 mmol L-1 for 10 min, followed by
the remaining activity determination. The activity without EDTA or metal ions
treatment was used as the control (100%).
The above procedure was also used to determine the effects of chemical agents
(Triton X-100, SDS, tween20 tween80 and DMSO) and organic reagents (methanol,
11

ethanol, isopropanol and ethyl acetate). The activity in the absence of any additives
was used as the control (100%).
All determinations were performed based on three replicates. R version 3.1.1
(Vanderbilt University, USA) was used for the statistical analysis. The one-way
ANOVA test was used, and a p value < 0.05 was deemed significant.
2.7 Whole cell catalysis for NA production.
The cells of E. coli BL21 (DE3-pET-nitA) and BL21 (DE3-pET-nitA-C2)
were used to carry out the whole cell catalysis experiments. The cell suspension of
10 g L-1 WCW was incubated in 100 mmol L-1 PBS buffer (pH 7.0) supplemented
with 100 mmol L-1 3-cyanopyridineat 37℃for 10 min. The reaction was terminated
by adding 100 μL HCl (2 mol L-1). Then, the cells were removed from the reaction
mixture by centrifugation at 8085 ×g and 4℃for 5 min. The supernatant was
diluted 10 times and filtered through a 0.22-μm-pore-size Millipore membrane for
HPLC analysis.
The concentrations of 3-cyanopyridine and NA were analyzed by High
Performance Liquid Chromatography (HPLC) with the Thermo Scientific™
Acclaim™ 120 C18 as the separation column (internal diameter, 4.6 mm; length,
250 mm). Samples were filtered through a 0.22 μm membrane before injection. The
mobile phase was acetonitrile: PBS buffer (pH 2.5) (15:85, vol/vol). The flow rate
12

was 1.0 mL min-1 and the working temperature was controlled at 25℃. The
detection wavelength was set at 217 nm and the injection volume was 20 μL.
Relative activity was calculated by the following equation: relative activity
(%) = specific activity detected / specific activity of the control enzyme *100%.
3. Results and Discussion
3.1 Sequence analysis.
The amino acids sequence of NitA shared 20-35% sequence that is identical
with the nitrilases from Syechocystis sp. PCC6803 (PDB accession No. 3WUY),
Agrobacterium sp. KNK712 (PDB accession No. 1ERZ) and Pyrococcus horikoshii
(PDB accession No. 1J31). The secondary structure (Fig. 1) analysis indicated that
NitA consisted of 11 β-strands and 11 α-helixes with a Glu48-Lys130-Cys164 triad
which is responsible for hydrolyzing non-peptide carbon-nitrogen bonds [23]. Since
the nitrilase from Syechocystis sp. PCC6803 (PDB accession No. 3WUY) shared
the highest sequence similarity (35%) with NitA, its crystal structure was used for
homology modeling of NitA by MOE software. The molecular docking studies
showed that the substrate, 3-cyanopyridine, interacts with Glu48-Lys130-Cys164
catalytic triad of NitA which indicated that the NitA might be able to catalyze the
conversion of 3-cyanopyridine into NA. Furthermore, sited-directed mutation
aimed at improving the catalytic activity of NitA towards 3-cyanopyridine was
conducted based on this homology structure. Wu et al. found that the Phe168 is a
13

significant site and the catalytic activity of Mut-F168V towards 1-
cyanocyclohexylacetonitrile was increased by 40-fold compared with wild type
protein [16,18,23]. Besides, Ser192, located on the flexible loop in the binding
cleft, was identified as another important region for altering enzyme activity
through docking experiments. Replacing S192 with bulkier Phe residues decreased
the size substrate-binding pocket and resulted in 1.8 and 1.9-fold improvement in
kcat/Km, respectively [17]. Thus these two sites were chosen for mutation and the
docking studies of the mutated NitA-C2 (F168V-S192F) showed that the distance
(3.99 Å) between the cyano group of 3-cyanopyridine and the sulfur of Cys of
mutated NitA-C2 was shortened compared with that of the wild-type enzyme
(5.04Å) (Fig. 2). This indicated that the substrate and the active pocket contacted
more closely in the NitA-C2 which might resulted in increased catalytic activity.
Based on above analysis, the original NitA and mutated NitA-C2 were constructed
and both were applied for NA production in this study.
3.2 Cloning, expression and purification of the NitA and NitA-C2.
As described in section 2, E. coli BL21(DE3) was used as the host cell and the
NitA and NitA-C2 (F168K-S192F) were successfully expressed as verified by the
SDS-PAGE analysis (Fig. 3) and specific activity assays (Table 2). Afterwards,
both nitrilases were purified by evolution through Ni2+-NTA resin and a single
band with approximately 45.2 kDa on the SDS-PAGE gel was observed for both
purified NitA and NitA-C2, which was in good agreement with the theoretical
14

molecular mass (Fig. 3). For NitA, a 53% recovery with 10-fold increase of specific
activity was obtained after purification. As to NitA-C2, the recovery rate was
decreased to 29% while the specific activity increased 8-fold to 35 U mg-1 protein
(Table 2). Moreover, the specific activity of purified NitA-C2 for 3-cyanopyridine
was 5-fold higher compared with that of purified wild NitA. Chauhan et al.
reported that the NitA could be purified via ammonium sulfate precipitation, anion
exchange and gel filtration and a 65% recovery rate was obtained [24]. Although
higher recovery rate was achieved by the method reported by Chauhan, the method
used in this study was more convenient and practical since only one step was
needed during the purification.
3.3 Biochemical properties of purified NitA and NitA-C2.
To further characterize biochemical properties of purified NitA and Nit-C2,
substrate specificity, enzymatic kinetics and effects of pH and temperature on
enzyme activities were conducted.
3.3.1 Substrate specificity. The substrate specificities of the NitA and NitA-C2
were determined using a broad substrate spectrum including aliphatic nitriles,
heterocyclic nitriles and aromatic mononitriles. As shown in Table 3, both nitrilases
exhibited catalytic activities towards aliphatic dinitriles (iminodiacetonitrile,
fumarodinitrile, 2-methylglutaronitrile, succinonitrile) and aromatic mononitriles
(3-cyanopyridine and 4-chlorobenzonitrile), whereas the specific activities towards
mandelonitrile and 3-phenylethylcyanide were not detected. Furthermore, higher
15

catalytic activities for aliphatic dinitriles than aromatic mononitriles were observed
for both nitrilases which were in agreement with the results reported [25]. When
comparing the specific activity of NitA-C2 and NitA towards different substrates, it
was found that the NitA-C2 exhibited improved catalytic activity for 3-
cyanopyridine and 4-chlorobenzonitrile while decreased catalytic activity for
succinonitrile and 2-methylglutaronitrile. As to aliphatic dinitriles, no significant
differences were observed.
The nitrilase from A. facilis had been reported to hydrolyze a wide variety of
nitriles and presented a strong bias for C3-C6 dinitriles over mononitriles of the
same chain length [21,22,25]. However, the mutant nitrilase (F168K-S192F)
constructed in this study possessed a higher activity towards 3-cyanopyridine which
is a pyridine ring with nitrile grouping (Table 3). Previous researches also showed
that the substrate specificity might be shifted after protein engineering. For
example, a mutated nitrilase from Rhodococcus rhodochrous ATCC 33278 with
non-polar aliphatic amino acids at position 142 showed increased activities towards
aromatic substrates and decreased activities towards aliphatic substrates [26].
Furthermore, a mutant nitrilase (W146Y) from Syechocystis sp. PCC68039
exhibited increased activities on benzonitrile, 2-cyanopyridine and 3-cyanopyridine
while decreased activities on fumaronitrile and 2-butenenitrile. Further structure
analysis by MD simulation demonstrated that W146 played an important role to
stabilize the hydrogen bond network around the active site and a small change of
16

the local environment by amino acid alteration could significantly change the local
coordination of the catalytic triad and the substrate binding location which
subsequently changed the enzymatic activity and substrate specificity [27].
3.3.2 Enzyme kinetics.
The kinetic parameters of the recombinant NitA and NitA-C2 for 3-
cynanopyridine were analyzed using a Lineweaver-Burk plot. The values of kcat, Km
and Vmax were calculated to be 17 s−1, 28 mmol L-1 and 23 μmol mg-1 min-1 for NitA
and 79 s−1, 36 mmol L-1 and 105 μmol mg-1 min-1 for NitA-C2, respectively.
Interestingly, it was found that the catalytic activity of NitA was severely inhibited
when the substrate concentration reached above 40 mmol L-1 while the inhibition
substrate concentration for NitA-C2 was 60 mmol L-1 (Fig. 4). These results
indicated that the NitA-C2 has better substrate tolerance than that of NitA.
Substrate inhibition is a common phenomenon observed during the enzymatic
hydrolysis for products synthesis. For enzymatic NA production, it was reported
that the conversion rate of 3-cyanopyridine by Nocardia globerula NHB-2 was
increased when the substrate concentration was raised from 10 mmol L-1 to 50
mmol L-1 while the catalytic efficiency began to decline gradually when substrate
concentration reached above 50 mmol L-1 [8]. Similarly, the catalytic efficiency of
E. coli JM109 harboring nitrilase for 3-cyanopyridine conversion reached the
highest rate at a substrate concentration of 25 mmol L-1 while catalytic efficiency
slowed down as the substrate concentration further increased [15]. Compared with
17

above researches, the Nit-C2 used in this study showed improved substrate
tolerance which is a good character in view of industrial application.
3.3.3 Effects of temperature on NitA and NitA-C2 activity and stability.
The optimal catalytic temperature for wild and mutant nitrilase was
determined with temperature ranging from 30℃to 80℃. As shown in Fig. 5A, the
optimal temperature for both nitrilases was 60℃while NitA-C2 exhibited higher
activity than NitA between 50℃and 60℃. Moreover, the specific activities of both
nitrilases decreased rapidly at a temperature < 40℃or > 65℃, and almost no
activity could be detected at a temperature > 80℃.These results indicated that the
nitrilase from strain 72W is a mesophilic enzyme.
The thermal stability of the purified enzyme was evaluated by incubating the
wild and mutated nitrilase at different temperatures ranging from 40℃to 60℃. As
shown in Fig. 5B, the NitA and NitA-C2 were stable and retained at least 80%
residual activity after 60 min at 40℃. However, further increase of temperature
resulted in decreased thermal stability for both NitA and NitA-C2 and the thermal
stability of NitA was better than that of NitA-C2. When conducted at 60℃, the
specific activity of NitA was completely lost after 60 min while the specific activity
of NItA-C2 was completely lost after almost 10 min. These results illustrated that
although the specific activity of NitA-C2 improved significantly, its
thermal stability was not as good as that of the wild protein.
18

3.3.4 Effects of pH on NitA and NitA-C2 activity and stability.
The effects of pH on the activities of both nitrilases were also determined. As
shown in Fig. 4, both nitrilases exhibited high catalytic activities at a pH range
from 6.0 to 8.0, and the optimum pH was 6.5. Further acidification or alkalinization
of the medium decreased the enzyme activities rapidly (Fig. 5C). In particular, the
activity was quite low in Gly-NaOH buffer which might be attributed to the
inhibitory effect of Gly-NaOH. As to the pH stability, the mutated NitA-C2 was
more sensitive to pH which was indicated by the decreased activity than that of
NitA at different pH (Fig. 5D).
3.3.5 Effects of metal ions and chemical agents on NitA and NitA-C2 activity.
As listed in Table 4, EDTA almost did not inhibit the enzymatic activity at a final
concentration of 1 mmol L-1, and the results indicated that the NitA represented
metal ion non-dependent nitrilase. Furthermore, the effects of metal ions and
chemical agents on NitA and NitA-C2 activities were compared. Among which,
Fe2+,Cu2+, Ni2+, Zn2+, Co2+, Ca2+,Ba2+ and Mg2+ ions had little effects on the
activity, whereas the activity of NitA and NitA-C2 increased up to 109% and 106%
under 1 mmol L-1 Mn2+.
In terms of the effects of various chemical agents on the enzyme activities (Table
5), the organic solvents (ethanol, methanol, isopropanol, ethyl acetate and DMSO)
strongly inhibited the enzymes activities except a relative weak inhibition effect for
19

methanol on NitA. Besides, the activities of both enzymes were decreased under the
existence of different surfactants except tween 20. These data suggested that the
nitrilase from strain 72W was a metal ion non-dependent enzyme but with poor
organic solvent tolerance.
3.4 Homology modeling and docking study
To investigate the potential mechanism for decreased pH and thermal stability of
NitA-C2, the 3D structures of NitA and NitA-C2 were compared and the pocket
which combined the substrates and the active site were enlarged after mutation (Fig
6C and D). Generally, hydrophobic core packing contributes to the pH and thermal
stability of a protein [29] and the enlargement of the pocket would result in the
exposure of the hydrophobic core with decreased stability. Thus we speculated that
the decreased pH and thermal stability might be attributed to the exposure of the
hydrophobic core caused by the enlargement of the pocket after mutation.
3.4 NA Production.
During the biological NA synthesis process, whole cell catalysis rather than
purified enzyme was preferred due to the advantage of easy operating [14,15]. Thus,
E. coli BL21 (DE3-pET-nitA) and BL21 (DE3-pET-nitA-C2) were collected for
transforming 3-cyanopyridine to NA after aerobic induction. The 3-cyanopyridine and
NA concentration were analyzed by HPLC and the retention time of 3-cyanopyridine
and NA were 7.89 min and 2.88 min, respectively (Fig. 7 A and B). For E. coli BL21
20

(DE3-pET-nitA), a 100% conversion of 0.1 mol L-1 3-cyanopyridine to NA was
achieved within 30 min at a cell mass concentration (fresh weight) of 10 mg mL-1 (0.3
mmol min-1 g-1 wet cell weight). By contrast, 0.1 mol L-1 3-cyanopyridine was
completely consumed within 10 min using E. coli BL21 (DE3-pET-nitA-C2) and the
conversion rate increased 2.3-fold to 1.0 mmol min-1 g-1 wet cell weight. A conversion
rate of 10 μmol min-1 g-1 wcw was obtained by using immobilized E. coli JM109
harboring the nitrilase gene from A. faecalis MTCC 126 [15]. Furthermore, 206 g g-1
dcw nicotinic acid with a conversion rate of 1.0 mmol min-1 g-1 dcw was achieved by
reutilization of immobilized Gibberella intermedia CA3-1with up to 28 batches [30].
Compared with these researches, the mutated NitA-C2 exhibited higher conversion
rate but the final NA concentration was still not comparable. Thus more efforts will
be needed to improve the whole process efficiency and the researches aimed at
improving the thermal and pH stability of NitA-C2 were also needed.
4. Conclusion
In this study, a nitrilase from A. facilis 72W was cloned and mutated for
transforming 3-cyanopyridine into NA. The specific activity of purified NitA-C2
(35 U mg-1 protein) increased by 5-fold compared with that of purified NitA (7 U
mg-1 protein) at the expense of decreased thermal and pH stability. Furthermore,
both NitA and NitA-C2 exhibited metal ion independency and mesophilic
characteristics with poor organic solvent tolerance. Finally, a conversion rate of 1.0
21

mmol min-1 g-1 wcw was achieved when using whole cell catalysis of BL21 (DE3-
pET-nitA-C2) which holds potential applications in biological production of NA.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (No. 21390200), a PH.D. Programs Foundation of Ministry of Education of
China (No. 20123221110008), the Key Science and Technology Project of Jiangsu
Province (BE 2016389), the Jiangsu Province Natural Science Foundation for
Youths (No. BK20140940), a Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions, Program for Changjiang
Scholars and Innovative Research Team in University (No. 06-A-047).
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Figure captions
Fig. 1. Sequence alignment of NitA with other nitrilases. β-Sheets are shown as
arrows in yellow, whereas α-helices are shown as bars in red. Residues involved in
enzymatic catalysis are highlighted in red rectangle. ‘*’ denotes residues that are
identical in all sequences, ‘:’ denotes conserved substations and ‘.’ indicates semi-
conserved substitutions. NitA was aligned with the following proteins (accession
numbers): Nit6803 (3WUY), D-NAase (1ERZ), RrNit (Q03217), HP0642 (LJ31).
26

Fig. 2. Docking analysis of nitrilases with 3-cyanopyridine. NitA (A) and NitA-C2
(B).
27

Fig. 3. SDS-PAGE analysis of the recombinant NitA and NitA-C2. M1: Premixed
Protein Marker (High); Lane 1: total soluble proteins from induced BL21(DE3)
harboring pET-28a(+); Lane 2: total soluble proteins from induced BL21(DE3)
harboring pET-nitA; Lane 3: purified total soluble proteins from induced BL21(DE3)
harboring pET-nitA; Lane 4: total soluble proteins from induced BL21(DE3)
harboring pET-nitA-C2; Lane 5: purified total soluble proteins from induced
BL21(DE3) harboring pET-nitA-C2; M2: Premixed Protein Marker (Low).
28

Fig. 4. Effects of the substrate concentration on the NitA and NitA-C2 activity.
Reactions were performed at 60℃ for 10 min with different substrate concentrations
(20-200 mmol L-1).
29