Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10

Article abstract of DOI:10.1021/jf202746a

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg²⁺ stimulated hydrolytic activity, whereas Cu²⁺, Co²⁺, Ni²⁺, Ag⁺, and Hg²⁺ showed a strong inhibitory effect. The K_m and v_max for mandelonitrile were 4.74 mM and 15.85 μmol min^-1 mg^-1 protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.

Full text of DOI:10.1021/jf202746a

ARTICLE
pubs.acs.org/JAFC
Gene Cloning, Expression, and Characterization of a Nitrilase from
Alcaligenes faecalis ZJUTB10†
Zhi-Qiang Liu,‡,§ Li-Zhu Dong,‡,§ Feng Cheng,‡,§ Ya-Ping Xue,‡,§ Yuan-Shan Wang,‡,§ Jie-Nv Ding,‡,§
Yu-Guo Zheng,*,‡,§ and Yin-Chu Shen‡,§
‡Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China
§Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology,
Hangzhou 310014, People’s Republic of China
ABSTRACT: Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids.
In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a
catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The
substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The
optimum pH and temperature for the purified nitrilase was 7ꢀ8 and 40 °C, respectively. Mg2+ stimulated hydrolytic activity,
whereas Cu2+, Co2+, Ni2+, Ag+, and Hg2+ showed a strong inhibitory effect. The Km and vmax for mandelonitrile were 4.74 mM and
15.85 μmol minꢀ1 mgꢀ1 protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of
20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(ꢀ)-mandelic acid was >99%. Characteristics
investigation indicates that this nitrilase is promising in catalysis applications.
KEYWORDS: Nitrilase, Alcaligenes faecalis, structure modeling, characterization, enantioselective hydrolysis
’ INTRODUCTION
different kinds of nitrilases.14 Recently, screening new nitrilases
and exploring new applications are becoming popular research
activities.
Recently, environmental concerns are rising because of waste
accumulation and limited supplies of petroleum feedstocks,
which has resulted in a higher demand for advanced enzymatic
or green approaches to perform efficient chemical reactions.1
Several enzymes have been exploited to perform stereo- and
regioselective chemical transformations to prepare kinds of
molecules useful for applications in the foodstuff, pharmaceuti-
cal, and chemical industries.2,3
In our previous work, a strain of Alcaligenes faecalis ZJUTB10
with a high nitrilase activity toward the conversion of racemic
mandelonitrile to (R)-(ꢀ)-mandelic acid was isolated and char-
acterized. The nitrilase specific activity reached 350.8 U/g and
the highest production rate was 9.3 mmol hꢀ1 gꢀ1 16
From its
.
high enzymatic activity and tolerance to a high concentration of
products, this strain appears promising for potential applications
in industry to produce carboxylic acids from nitriles.16
The objectives of the research were to clone and express the
gene encoding nitrilase from A. faecalis ZJUTB10 as well as
perform an investigation of characteristics for purified nitrilase
and a prediction of the three-dimensional (3D) structure model
based on sequence similarity. These primary features suggest that
the A. faecalis nitrilase is a suitable enzyme for degradation or
modification of nitriles by the biocatalysis route.
Nitrilase (EC 3.5.5.1), as an important industrial enzyme, is
a valuable biocatalyst that hydrolyzes nonpeptide carbonꢀ
nitrogen bonds. Nitrilase belongs to the nitrilase family of CN-
hydrolyzing enzymes,4 which has drawn substantial interest as a
valuable alternative to chemical hydrolysis in organic chemical
processes and detoxification of cyanide wastes.5ꢀ7 In addition,
recently nitrilase has been widely used in the preparation of
pharmaceutical intermediates and phytocides and in the textile
industry.8,9 Nitrilases are preferred over chemical methods
because they can convert nitriles directly into the corresponding
carboxylic acids in a single step, are often used under ambient
conditions, and do not result in large amounts of byproducts.10
Nitrilase has the remarkable ability to readily facilitate chemical
hydrolysis processes and thus represents an excellent tool to
perform detailed structureꢀactivity analysis. Studies on nitrilases
have been widely carried out in terms of their occurrences,
applicabilities, catalytic mechanisms, and cloning of the nitrilase
genes from different origins.11ꢀ13 On the basis of the substrate
specificity, nitrilases are classified into three major categories
including aromatic nitrilases, aliphatic nitrilases, and arylacetoni-
trilases, making them useful for the hydrolysis of a large number
of nitriles.14 Nitrilases have been found in many plants and
microorganisms.15 A variety of nitriles have been transformed by
’ MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Culture Conditions.
A. faecalis ZJUTB10, grown in a medium that consisted of ammonium
acetate 12.14 g/L, yeast extract 7.79 g/L, K2HPO4, 5 g/L, MgSO4 0.2 g/L,
NaCl 1 g/L, and n-butyronitrile 3.29 g/L, pH 7.5, has been deposited at
the China Center for Type Culture Collection (CCTCC M 208168,
Wuhan, China).16 The Escherichia coli strains JM109 (Tiangen biotech
Co., Ltd., Beijing, China) and BL21 (DE3) (Invitrogen, Karlsruhe,
Received: July 9, 2011
Accepted: September 13, 2011
Revised:
August 31, 2011
Published: September 13, 2011
r
2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
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Germany) were used as hosts for cloning and expression experiments,
respectively. The plasmids pMD18-T (TaKaRa, Otsu, Japan) and pET-
28b(+) (Novagen, Darmstadt, Germany), carrying an N-terminal
and a C-terminal 6ꢁHis-Tag sequence, were used for cloning and
expression of nitrilase, respectively. E. coli and recombinant E. coli strains
were grown in LuriaꢀBertani (LB) (yeast extract 5 g/L, tryptone 10 g/L,
NaCl 5 g/L) liquid medium at 37 °C. To maintain the presence of the
plasmids, ampicillin and kanamycin were added to the media for E. coli
harboring plasmids pMD18-T and pET28b(+), respectively.
previously equilibrated with a binding buffer (20 mM NaH2PO4, pH 8.0;
300 mM NaCl). Unbound proteins were washed out from the column
with a washing buffer (20 mM NaH2PO4, pH 8.0; 300 mM NaCl;
50 mM imidazole). Then the elution was carried out with a buffer
(20 mM NaH2PO4, pH 8.0; 300 mM NaCl; 500 mM imidazole). The
proteins were analyzed using 12% sodium dodecyl sulfate polyacryla-
mide gel electrophoresis (SDS-PAGE).
Construction of Nitrilase Mutants. Site-directed mutants of the
nitrilase at positions of Glu-47, Lys-129, and Cys-163 were constructed
using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA) and pET28b(+)-NIT plasmid as a template. Glu 47 was
mutated to Gln with primers Glu47Gln (F): 50-GATCGTGTTCGGT-
CAGACTTGGCTG-30, and Glu47Gln (R): 50-GTCTGACCGAA-
CACGATCAGATCAC-30. Lys 129 was mutated to Ile with primers
Lys129Ile (F): 50-TGTGGTCCCGTCGTATACTGAAACC-30, and
Lys129Ile (R): 50-CAGTATACGACGGGACCACAGCATTTC-30.
Cys 163 was mutated to Gly with primers Cys163Gly (F): 50-
TGTCGGCGCTCTGTGTGGCTGGGAACATCT-30, and Cys-
163Gly (R): 50-AGCCACACAGAGCGCCGACACGGCCCAGT-30.
Mutants were verified by sequencing, expressed, and purified in the
same way as that used for the wild type enzyme.
SDS-PAGE Analysis. Crude extracts and elution fractions were
analyzed by SDS-PAGE, performed using a Mini-gel system (Bio-Rad).
The gels were cast with 0.75 mm spacers (Bio-Rad). Aliquots of each
eluted fraction were subjected to SDS-PAGE and enzyme assay. SDS-
PAGE was performed according to Laemmli’s discontinuous Tris-
glycine buffer system at 10 mA for 2ꢀ3 h with a 5% acrylamide stacking
gel (pH 6.8) and 12% separating gel (pH 8.8).18 Proteins in the gel were
stained with Coomassie brilliant blue R-250.
Enzyme Assays and Protein Assay. The activity of nitrilase was
determined by measuring the production of (R)-(ꢀ)-mandelic acid
from mandelonitrile.16 The standard assay was performed with mande-
lonitrile (20 mM) and the enzyme mixed in Tris-HCl buffer (50 mM,
pH 7.5). The reaction mixture (total volume 5 mL) was incubated at
40 °C, 150 rpm. After 10 min, 500 μL of mixture was sampled, 50 μL of
HCl (2 M) was added to end the reaction, and then the mixture was
centrifuged at 12 000g for 2 min under 4 °C. The amount of mandelic
acid product was determined by a LC-20AD prominence liquid chro-
matography instrument (Shimadzu, Kyoto, Japan) using a C18 column
(5 μm ꢁ 250 nm ꢁ 4.6 nm, Elite Analytical Instruments Co., Ltd.,
Dalian, China) and equipped with a SPD-2A prominence UV/vis
detector. Each sample (20 μL) was eluted at 30 °C with 0.01 M
(NH4)H2PO4:CH3OH = 13:7 (v/v, 1 mL/min) as mobile phase, and
A228 was measured.
Gene Manipulation. DNA manipulation, plasmid isolation, and
agarose gel electrophoresis were conducted according to standard
protocol 17 unless stated otherwise.
Construction of pMD18-NIT Cloning Vector. The genomic
DNA of A. faecalis ZJUTB10 was used as the template, and the open
reading frame of the nitrilase gene was amplified by PCR. The primers
that were designed based on the reported amino acid sequences of
nitrilases in NCBI (Accession Nos. P20960 and CAC09069) for the
amplification of genes NIT were P1: 50-ATGCAGACTCGTAAA-
ATTGTT-30 and P2: 50- GGACGGTTCCTGAACCAG-30. The PCR
reaction mixture consisted of 20 ng of genomic DNA, 50 μM for each
dNTP, 0.5 μM P1 and P2, 5 μL of 10ꢁPfu DNA buffer, and 2 U Pfu
DNA polymerase (Biocolor, Shanghai, China) in 50 μL. PCR was
carried out in 50 μL using a Thermocycler (Bio-Rad, Hercules, CA).
PCR products were visualized on a 1.5% ethidium bromide stained gel.
The reaction was carried out at 94 °C for 5 min, followed by 35 cycles at
94 °C for 45 s, 55 °C for 45 s for amplifying the nitrilase gene, 72 °C for
2 min, and then 72 °C for 10 min in a PCR thermocycler (Bio-Rad). An
approximate 1.1 kb fragment was subjected to 0.9% agarose electro-
phoresis and then purified (Axygen, Union City, NJ). Then the fragment
was cloned into the plasmid of pMD18-T (Takara, Otsu, Japan) by T/A
cloning and transformed into E. coli JM109 competent cells. Finally, the
positive clone was sequenced. 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. De-
duced amino acid sequences were obtained from ExPASy and GenBank
databases.
Construction of Expression Plasmid for Nitrilase Gene.
According to the sequence of the nitrilase gene of A. faecalis ZJUTB10, a
pair of primers was redesigned as follows: P3: 50-AATGGATCCATG-
CAGACTCGTAAAATTGTTCG-30 and P4: 50-AGGGTCGACG-
GACGGTTCCTGAACCAGC-30. The PCR products were double
digested with BamHI and SalI. The resulting 1.1 kb fragments containing
the intact nitrilase genes were inserted into pET28b(+) vector which had
been linearized with the enzymes of BamHI and SalI using T4 ligase.
Transformation of expression plasmids pET28b(+)-NIT into E. coli
BL21 (DE3) was performed by the heat-shock method.
Expression and Purification of Recombinant Nitrilase. The
recombinant plasmids carrying the wild type and mutant nitrilase genes
were transformed into E. coli BL21(DE3) cells. The resulting recombi-
nant cells were grown at 37 °C in 50 mL of LB media containing 50 μL of
kanamycin (50 mg/mL). Production of enzyme was induced by addition
of 0.1 mM isopropyl β-D-thiogalactoside (IPTG) when optical density at
600 nm of the culture broth reached between 0.6 and 0.8. The cells were
then incubated at 28 °C for 20 h and harvested by centrifugation at
9000 rpm for 20 min. The harvested cells were washed twice with 10 mL
of physiological saline solution (NaCl 0.9%). Then the cells were
resuspended in 30 mL of 50 mM Tris-HCl (pH 7.5) and lysed by
sonication with a Vibra-Cell VC 505 ultrasonic processor (Sonics and
Materials Inc., Newtown, CT) at 350 W for 30 min. The soluble
fractions of the sonified solution were obtained by centrifugation at
9000 rpm for 20 min.
The optical purity of (R)-(ꢀ)-mandelic acid was determined by
analysis of the enantiomers on a CHIRALEL-OD-H column (250 ꢁ 4.6
mm) (Daicel Chemical Industries, Shinzaike, Japan) at a flow rate of
0.8 mL/min with a mobile phase containing hexane, 2-propanol, and
trifluoroacetic acid (90:10:0.1, v/v) at A228. Before optical purity
determination, pretreatment steps were performed on the reaction
mixture. The pH of the reaction mixture was adjusted to about 1.5,
and then the enantiomers were extracted with equal volume of ethyl
acetate. The extract obtained was concentrated, and the residual solid
was dissolved in the mobile phase for HPLC determination. The ee of
(R)-(ꢀ)-mandelic acid was calculated by the eq 1:
ee ¼ ½ðR ꢀ SÞ=ðR þ SÞꢂ ꢁ 100%
ð1Þ
where R and S represent the concentration of (R)-(ꢀ)-mandelic acid
and (S)-(+)-mandelic acid, respectively.
One unit of the enzyme activity was defined as the amount of enzyme
that produces 1 μmol of mandelic acid per minute under the conditions
described above. Protein quantitative analysis was conducted using the
Bradford method19 with bovine serum albumin as a standard.
The resulting crude extracts were retained for purification. The cell-
free extracts were applied onto a nickel-NTA superflow column (10 mL)
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Table 1. Characteristics of Some Nitrilases
organism
substrate
molecular mass (kDa)
optimal pH
optimal temp (°C)
reference
this work
27
Alcaligenes faecalis
mandelonitrile
benzonitrile
44.2
38.5
7ꢀ8
40
45
Aspergillus niger K10
4-cyanophridine
benzonitrile
8
Geobacillus pallidus DAC521
crotonitrile
41
7.6
8
65
30
45
65
45
50
45
45
28
Norcadia (Rhodoccocus) NCIMB 11216
Rhodococcus rhodochrous J1
benzonitrile
m-nitrobenzonitrile
benzonitrile
45.8
40
29,30
31,32
33,34
35
acrylonitrile
7.6
8ꢀ9
7ꢀ8
5.5
7.5
7.5
Acidovorax facilis 72W
fumaronitrile
benzonitrile
40
Bradyrhizopium japonicas USD 110(blr3397)
Rhodococcus rhodochrous K22
hydrocinnamonitrile
benzonitrile
34.5
41
acrylonitrile
benzonitrile
36
Alcaligenes faecalis ATCC 8750
Alcaligenes faecalis JM3
p-aminobenzylcyanide
benzonitrile
32
37
2-thiopheneacetonitrile
p-chlorobenzylcyanide
phenylacetonitrile
mandelonitrile
KCN
38.9
38,39
Bradyrhizopium japonicas USD 110(bll6402)
37
38
45
/
/
40
41
42
Pseudomonas stutzeri AK61
7.5
7.5
30
25
Fusarium solani
KCN, K2Ni(CN)4, K4Fe(CN)6
Time Course of Mandelonitrile Hydrolysis. The time course
of mandelonitrile hydrolysis was examined with 20 mM mandelonitrile
and the appropriate amount of enzyme under standard conditions.
Samples were periodically withdrawn from separate reaction mixtures
under identical conditions, and the concentrations of mandelonitrile,
(R)-(ꢀ)-mandelic acid, and (S)-(+)-mandelic acid were determined.
Temperature and pH Dependence. The optimal temperature
of the enzyme was determined by assaying enzyme activities at tem-
peratures ranging from 25 °C to 65 °C. Thermostability was investigated
by incubating enzymes at temperatures ranging from 35 °C to 65 °C at a
final concentration of 0.1 mg/mL in 50 mM Tris-HCl buffer (pH 7.5).
The incubation took place in a heat-block at the specified temperature
for 1 h. The enzyme activities were assayed at 40 °C after various
incubation times. To investigate the optimal pH and the effect of pH on
activity of nitrilase, three different buffers with concentrations of 50 mM
including potassium phosphate buffer (pH 6.0ꢀ7.5), Tris-HCl buffer
(pH 7.0ꢀ9.0), and glycineꢀsodium hydroxide buffer (pH 8.5ꢀ10.0)
were used, and reactions were performed at 40 °C using 20 mM
mandelonitrile as substrate. To investigate pH stability, the enzyme
was dissolved in different buffers of pHs ranging from 6.0 to 10.0 and
incubated at 40 °C for 1 h, and then the relative activities were carried
out in the standard enzyme assay condition. Activity without treatment
obtained at pH 7.5 was used as a blank and regarded as 100%. All the
experiments were performed in triplicate, and the data obtained are
presented as activity without normalization.
250 nm with a scan speed of 20 nm/min. Results are expressed in terms
of mean residue ellipticity ([θ]MRW,λ) (in deg cm2 d molꢀ1) determined
according to eq 2:20,21
½θꢂMRW, λ ¼ MRWðθλÞ=10lc
ð2Þ
where MRW is the mean residue weight of the specific cutinase, θλ is the
observed ellipticity (mdeg), l is the path length (cm), and c is the protein
concentration (μM). Thermal denaturation of enzymes was followed as
a function of temperature and pH by continuously monitoring ellipticity
changes at 222 nm using a step size of 1 °C. Observed ellipticities were
transformed to apparent fractions of denatured proteins (f
)
D,app
according to eq 3:
fD, app ¼ ðθT ꢀ θNÞ=ðθD ꢀ θNÞ
ð3Þ
where θT is the sample ellipticity (mdeg) at a particular temperature, and
θD and θN are the corresponding values for the denatured and native
states, extrapolated to the same temperatures.22 The Tm was calculated
by taking the first-order derivative of the sigmodial curve obtained from
the melting curve.23 These experiments were conducted in triplicate, and
averaged data are reported here.
Metal Ion and Chemical Analysis. The effects of metal ions and
chemicals on the enzyme activity were measured after the enzyme was
preincubated at 40 °C with various compounds at a final concentration
of 5 mM for 10 min in Tris-HCl buffer at pH 7.5. After treatment, the
activities were estimated according to the standard activity assay method
mentioned above.
Substrate Specificity. Enzyme activity toward nitriles was deter-
mined by either the formation of ammonia or the production of car-
boxylic acids. The reaction was performed in a reaction mixture (0.5 mL)
containing 10 mM nitrile, 50 mM pH 7.5 Tris-HCl, and an appropriate
amount of the enzyme at 40 °C for 10 min with a reciprocal shaker at
150 rpm (standard conditions). The amounts of o-methoxyphenylacetic
acid, m-methoxyphenylacetic acid, and p-methoxyphenylacetic acid were
Circular Dichorism (CD) Measurements. CD spectra were
recorded on a JASCO J-815 Spectropolarimeter (JASCO Inc., Easton,
MD) using Spectra Manager 228 software. The temperature was
controlled using a Fisher Isotemp Model 3016S water bath (Fisher
Scientific, Waltham, MA). A lyophilized powder (purity >95% w/w) was
prepared. A stock solution of 0.25 mM nitrilase was generated in 20 mM
sodium phosphate, pH 3.0, sodium acetate, pH 5.0, and 20 mM sodium
phosphate, pH 8.0. Far-UV scans were performed at 0.15 mg/mL
protein in a 10-mm cuvette. The spectra were recorded from 200 to
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Figure 1. Structure-based alignment of nitrilase from different origins. AF10, an arylacetonitrilase nitrilase from A. faecalis ZJUTB10; RRK22, an
aliphatic nitrilase from Rhodococcus rhodochrous K22 (D12583); RRJ1, an aromatic nitrilase from R. rhodochrous J1 (D11425); AN10, an aromatic
nitrilase from Aspergillus niger K10 (EU282000); RRNCIMB, an aromatic nitrilase from R. rhodochrous NCIMB 11216 (CAC88237); 72W, an aliphatic
nitrilase from Acidovorax facilis 72W (DQ444267); 1J31, hypothetical protein PH0642 (PDB accession code 1J31) one of the templates. The red color
indicates the conserved catalytic residues: The residues influencing the triad pocket (Cys-163, Glu-47, and Lys-129) are marked in green.
determined by HPLC (LC-10AS, Shimdazu, Kyoto, Japan) using a 4.6
mm ꢁ 250 mm ODS column (Shimadazu, Kyoto, Japan). The param-
eters used for the detection of the compounds were a UV detector set
at a wavelength of 270 nm, a flow rate of 1.0 mL/min, and a mobile phase
of water/acetonitrile (50:50, v/v) containing 0.1% trifluoroacetic acid.
The amounts of mandelic acid, o-chloromandelic acid, and phenylacetic
acid were determined by a LC-20AD prominence liquid chromatogra-
phy instrument (Shimadzu, Kyoto, Japan) using a C18 (5 μm ꢁ 250 nm
ꢁ4.6 nm) column (Elite Analytical Instruments Co., Ltd.) and equipped
with a SPD-2A prominence UV/vis detector. Each injected sample
(20 μL) was eluted at 30 °C with 10 mM (NH4)H2PO4:CH3OH =
13:7 (v/v, 1 mL/min) as mobile phase, and products were detected
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Figure 2. SDS-PAGE analysis of expression products by E. coli BL21-
(DE3) (pET-28b(+)-NIT). Gels with 30% polyacrylamide were sub-
jected to protein analysis, and the protein bands were stained with
Coomassie blue R250. M: Protein marker β-galactosidase (116.0 kDa),
bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehy-
drogenase (35.0 kDa), REase Bsp98I (20.1 kDa), β-lactoglobulin
(18.4 kDa), and lysozyme (14.4 kDa). Lanes 1 and 2 stand for the
products of E. coli BL21 (DE3) control and E. coli BL21 (DE3) [pET-
28b(+)-NIT] negative, lane 3 stands for induced E. coli BL21 (DE3)
[pET28b(+)-NIT], and lane 4 stands for purified nitrilase.
at 228 nm. A Hypersil SAX icon exchange column (Shimadazu, Kyoto,
Japan) was used for quantitative analysis of iminodiacetic acid. The
parameters used for the detection of the compounds were a UV detector
set at a wavelength of 210 nm, a flow rate of 1.0 mL/min, and a mobile
phase of 0.02 M (NH4)2HPO4 at pH 4.0. Concentrations of 2-amino-
2,3-dimethylsuccinic acid, 2,2-dimethylcyclopropanecarboxylic acid,
acrylic acid, benzoic acid, acetic acid, 4-aminobenzoic acid, aminobutyric
acid, and hexanedioic acid were assayed using an Agilent 6890N gas
chromatograph (Agilent, Santa Clara, CA), equipped with a flame
ionization detector (FID) and a FFAP column (30 m ꢁ 0.25 mm
ꢁ0.33 μm). The operating conditions were as follows: oven temperature
was 180 °C, and the injection and detector temperature were kept at
250 °C for analysis. One unit of the enzyme activity toward each
substrate was defined as the amount of enzyme required to produce
1 μmol of the corresponding acid per minute at 40 °C, respectively. The
activities were given relative to the activity toward mandelonitrile. The
activity toward mandelonitrile was considered as 100%.
Figure 3. Effect of temperature on activity of nitrilase and stability. (a)
Reactions were performed at 150 rpm for 10 min at various temperatures
(25 °C to 65 °C) with 50 mM Tris-HCl buffer (pH 8.0). (b) The
enzyme was preincubated at 40 °C and reacted for 10 min.
with pMD18-T, and the recombinant plasmid, named pMD18-
T-NIT, was transformed into a E. coli JM109 competent cell by
heat shock. A number of positive clones (colorless) with
ampicillin resistance were obtained. Among these, three were
picked to prepare the single-strand for sequence analysis. As a
result, a sequence of about 1.1 kb was achieved. The sequence
analysis using software DNAMAN (Version 5.2, Lynnon Biosoft,
Quebec, Canada) indicated that the amplified fragment con-
tained an ORF of 1068 bp encoding 356-amino-acid protein with
a molecular mass of 41.5 kDa. The nitrilase gene sequence
obtained here was submitted to the GenBank database with
the accession number of HQ407378. The sequences alignment
result showed that nitrilase from A. faecalis ZJUTB10 showed a
high homology of about 78% with those of Pseudomonas putida
strain MTCC 5110 (GeneBank acession no. EF467660) and
Alcaligenes sp. ECU0401 (GeneBank acession no. FJ943638).
Although nitrilases have been well studied, the nitrilase obtained
in this study is quite different compared with the previously
reported nitrilases from different origins (Table 1) which share
the same conserved regions, including PGYP (residues 52ꢀ55),
RRKLKPTHVER (residues 128ꢀ138), CWEH (residues 164ꢀ
167), and GHYS (residues 295ꢀ298) (Figure 1).
Homology Modeling and Docking. The three-dimensional
homology model of nitrilase was generated using Build Homology
Models (MODELER) in Discovery Studio 2.1 (DS 2.1, Accelrys Soft-
ware, San Diego, CA) using crystal structures of a hypothetical protein
PH0642 (PDB accession code 1J31) and a thermoactive nitrilase (PDB
accession code 3IVZ) as templates. The generated structures were
improved by subsequent refinement of the loop conformations by
assessing the compatibility of an amino acid sequence to known PDB
structures using the Protein Health module in DS 2.1. The geometry of
loop regions was corrected using Refine Loop/MODELER. Finally, the
best quality model was chosen for further calculations, molecular
modeling, and docking studies by Autodock 4.0.24 Structure-based
sequence alignment was performed using the program ClustalX,25 and
the picture of the sequence alignment was made using the program
ESPRIPT.26
’ RESULTS
Cloning and Sequence Analysis of the Nitrilase. The
products amplified with the primers P1 and P2 were subjected
to agarose gel electrophoresis, and fragments of about 1.1 kb
were generated. The fragments were then recovered and ligated
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Figure 5. Effect of pH on activity of nitrilase and stability. (a) Effect of
pH on activity of nitrilase. Reactions were performed at 40 °C, 150 rpm,
for 10 min with different buffers. (b) pH stability: The purified enzyme
was incubated in different buffers at pHs ranging from 6.0 to 10.0 for 1 h,
and the residual activity was tested. All experiments were performed in
triplicate.
Figure 4. Spectroscopic structural analysis and thermal stability of
nitrilase. (a) CD wavelength scans of nitrilase at pH 7.5. The data are
represented as the average of three trials. (b) Far-UV CD spectra of
nitrilase from 44 °C to 64 °C. Relative activity and molar ellipticity were
measured at 222 nm.
Optimal Temperature and Thermostability. To evaluate
the optimal temperature of the enzyme, activities were assayed at
various temperatures ranging from 25 °C to 65 °C using standard
assay conditions, and detailed results are shown in Figure 3a. The
activity of nitrilase increased from 25 °C to 40 °C and reached its
maximum at a temperature of 40 °C which is similar to that of
nitrilase from P. putida.43 However, after this apex point, the
activity of nitrilase decreased sharply and the activity was totally
lost at 65 °C. To investigate the thermostatbility of nitrilase, the
enzyme was incubated at different temperatures ranging from
35 °C to 65 °C, and the activities of enzymes that were assayed
under the standard reaction conditions were used as control.
The activity decreased markedly above 55 °C, which might be
explained by the inactivation or changes in configuration of the
nitrilase while, like most of the nitrilases reported in literature,
the enzyme was labile at higher temperature as revealed by a half-
life of 57 min at 40 °C, 42 min at 45 °C, and 20 min at 55 °C,
respectively (Figure 3b). The enzyme thermostability was better
than that of nitrilase from P. putida, reported to have half-lives
of 76 min at 40 °C and 9 min at 50 °C, respectively.43 Indeed,
the enzyme seemed to be still sensitive to heat, which can be
corroborated by thermostability measurements with CD (Figure 4)
that A. faecalis nitrilase exhibited a melting temperature of 56 °C.44
Expression of the Nitrilase Gene and Its Purification. To
express the gene encoding nitrilase, primers P3 and P4 were
designed according to the sequencing result of pMD18-T-NIT,
with BamHI and SalI sites, respectively. PCR amplification was
conducted by adopting the recombined plasmid pMD18-T-NIT
obtained above as the template, and the DNA fragment encoding
the nitrilase gene was subcloned into an expression vector
pET28b(+) to construct the recombinant plasmid pET28b(+)-
NIT. Subsequently, the recombinant plasmids were then intro-
duced into competent cells of E. coli BL21(DE3), and many
clones were picked up. The positive transformant containing
recombinant pET28b(+)-NIT was identified by colony PCR and
double enzymatic digestion. The recombinant cell harboring
pET28b(+)-NIT was induced by 0.1 mM of IPTG at 28 °C for
about 20 h when the OD600 reached the value of 0.6. Protein
bands from SDS-PAGE were visualized by staining with Coo-
massie brilliant blue. The thick protein band corresponding to
the subunit of nitrilase with the 6ꢁHis-tag shows an apparent
molecular mass of about 44 kDa (Figure 2). These data are in
excellent agreement with those derived from DNA sequencing.
The purified protein was observed as a single band with SDS-
PAGE (Figure 2), indicating that the protein was quite pure.
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Table 2. Effect of Metal Ions and Some Chemicals on the
Activity of Nitrilase from A. faecalis ZJUTB10a
reagent (5 mM)
relative activity (%)
control
CuCl2
CoCl2
FeCl3
100
16
20
81
83
17
78
102
89
122
92
64
6
FeCl2
NiCl2
MnCl2
CaCl2
LiCl
MgCl2
BaCl2
ZnSO4
AgNO3
HgCl2
EDTAꢀNa2
Figure 6. Time course of mandelonitrile by the nitrilase. Reactions were
performed at 40 °C, 150 rpm. The purified enzyme (1 mg) was
incubated with 10 mL of 50 mM Tris-HCl buffer (pH 7.5) and
20 mM for each nitrile.
0
96
a Enzyme activities were determined under standard assay conditions, in
the presence of 5 mM of each salt and expressed as the percentage with
respect to the activity of a control reaction carried out in the absence of
any salt.
conversion rate of 100%. However, the nitrilase of P. fluorescens
EBC191 only produced 19% of the total products in terms of
the corresponding acid.45 These results indicate that a compli-
cated and costly downstream process can be avoided and
that a high concentration and purity of product can be attained,
thus reducing the operation cost of an enzymatic process
from the viewpoint of industrial application. The optical purity
of the (R)-(ꢀ)-mandelic acid product was maintained above
99% ee during the reaction process (Figure 6), which was the
same as that from using the strain A. faecalis ZJUTB10 as cata-
lyst (>99% ee).16
Optimal pH and pH Stability. To investigate the optimal pH,
buffers including potassium phosphate buffer (pH 6.0ꢀ7.5),
Tris-HCl buffer (pH 7.0ꢀ9.0), and Gly-NaOH buffer (pH
8.5ꢀ10.0) with 50 mM for each were used. The results showed
that the highest activity was observed in Tris-HCl at pH 7.5
though nitrilase functions in all the buffers (Figure 5a). The pH
stability of enzyme was examined by assaying the residual activity
under the standard assay conditions after incubation. The results
shown in Figure 5b suggested that the nitrilase was more stable at
pH 7.0 with about 65% residual activity. However, higher pH
(pH > 8.0) resulted in significant reduction in the residual activity
to approximately 20% (Figure 5b).
Effects of Metal Ions and Ethylenediaminetetraacetic Acid
(EDTA). The effects of different metal ions and EDTA on the
activity of nitrilase were examined by incubating the enzyme in
the presence of the reagents. The activity assayed in the absence
of metal ions and EDTA was considered as the control. The data
shown in Table 2 indicate that activity of nitrilase was affected by
most tested metal ions and was especially strongly inhibited by
Cu2+, Co2+, Ni2+, Ag+, and Hg2+. As we know, Ag+ and Hg2+
have strong binding to sulfhydryl reagents, which indicates that
the thiol group is essential for the catalytic activity. In addition,
the results of metal ions analysis showed that the Mg2+ could
slightly activate the activity of nitrilase. However, no inhibition
was observed in the presence of chelating agent EDTA, indicating
that this nitrilase had no metal ion requirements.
Determination of the Kinetic Parameters. For determin-
ing the kinetics of the hydrolysis reaction, the rate of this reaction
was determined at different concentrations [S] (0ꢀ60 mM) of
racemic mandelonitrile (Figure 7). The experimental data col-
lected were fitted to the MichalisꢀMenten equation (v0 =
(vmax[S]/2)/(km + [S]/2)) to determine the Km and vmax. The
Km and vmax of the nitrilase for (R)-(ꢀ)-mandelic acid were
calculated to be 4.74 mM and 15.85 μmol minꢀ1 mgꢀ1
,
respectively.
Substrate Specificity. The nitrilase from A. faecalis ZJUTB10
was assessed for hydrolytic activity on a set of nitriles listed in
Table 3. The purified enzyme has no activity toward aminobu-
tytonitrile, 2,2-dimethylcyclopropyl cyanide, and 2-amino-2,
3-dimethylbutanenitrile, very low activity toward 4-aminobenzo-
nitrile and iminodiacetonitrile, but high activity toward pheny-
lacetonitrile, o-methoxyphenylacetic acid, m-methoxyphenyla-
cetic acid, and p-methoxyphenylacetic acid. From the substrate
specificity study, though the enzyme exhibited activity toward
both classes of aromatic and arylacetonitriles, we found that the
nitrilase showed higher activity toward the arylacetonitriles,
which further confirmed that A. faecalis nitrilase obtained here
belongs to the arylacetonitrilase family.
Homology Modeling and Docking. The three-dimensional
homology models of nitrilases were generated using Build
Homology Models (MODELER) module in Discovery Studio
2.1 (Accelrys Software). The crystal structure of hypothetical
protein PH0642 (PDB accession code 1J31, resolution 1.6 Å)
from Pyrococcus horikoshii,46 and a thermoactive nitrilase PaNit
(PDB accession code 3IVZ, resolution 1.57 Å) from P. abyssi,47
Time Course of Mandelonitrile Hydrolysis with Recombi-
nant Nitrilase. The data shown in Figure 6 indicate that the
R-isomer was rapidly transformed to acid, which was much
faster than that for the S-counterpart. After 35 min, the concen-
tration of (R)-(ꢀ)-mandelic acid in the reaction mixture
reached approximately 20 mM by adding 1 mg of purified
enzyme in 10 mL of Tris-HCl buffer at pH 7.5. Because of the
efficient in situ racemization of the unreacted (S)-mandelonitrile
at slightly alkaline pH,37 we did not detect any amount of
mandelamide as a possible byproduct, resulting in a substrate
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Figure 7. Reaction rate at different mandelonitrile concentrations.
Reactions were performed for 10 min at 40 °C, 150 rpm, with 50 mM
Figure 8. View of modeled structure of A. faecalis ZJUTB10 nitrilase.
Tris-HCl buffer (pH 7.5): ( ) fitting curve, (—) experimental curve.
3 3 3
improved by subsequent refinement of the loop conformations
by assessing the compatibility of an amino acid sequence to
known PDB structures using the Protein Health module in DS
2.1. The geometry of loop regions was corrected using Refine
Loop/MODELER. Finally, the best quality model (Figure 8) was
chosen for further calculations, molecular modeling, and docking
studies. The conserved catalytic residues Glu-47, Lys-129, and
Cys-163 from nitrilase protein were proposed to be the catalytic
triad of nitrilase and have similar orientations and locations in the
nitrilase model. To verify the prediction of the conserved
catalytic residues, and test whether the predicted conserved
catalytic residues play a role in catalysis, these residues were
mutated by site-directed mutagenesis. The mutations of these
residues resulted in inactivated enzymes, the activities being
hardly detectable, indicating that these residues are involved in
enzymatic activity. However, the accuracy of the model is limited,
as it is based on homology modeling and not on a crystal
structure. To confirm our molecular modeling and structural
analysis, the crystal structure of A. faecalis ZJUTB10 nitrilase
should be determined.
The molecular docking studies of the A. faecalis ZJUTB10
nitrilase show substrate interactions in the active site. The
resulting model was used for further docking studies. Hydrogen
atoms were added to the protein model. The added hydrogen
atoms were minimized to have a stable energy conformation and
also to relax the conformation from close contacts. The active site
of nitrilase was defined, and a cubic of 40 Å3 was generated
around the catalytic triad pocket (CEK). With the AD4 force
field, 53 substrates (Table 4) were able to be docked in the
receptors (enzymes) in productive conformations. The docked
energy (10 docked conformations) of each substrate/enzyme
pair was compared, and the results are shown in Table 3. In the
catalytic triad, Cys-163 attacks the cyano group of the nitriles as a
nucleophile, Glu-47 assumes the role of a general base, and Lys-
129 is involved in the stabilization of a tetrahedral intermediate
structure, which are the same features as those observed for the
nitrilase from P. fluorescens Pf-5.48
Table 3. Substrate Specificity of the Nitrilasea
docked energy
substrate
relative activity (%)
(kcal/mol)
(R)-mandelonitrile
iminodiacetonitrile
acetonitrile
100
12
36
0
ꢀ2.664
ꢀ2.473
ꢀ2.488
ꢀ1.783
ꢀ2.434
ꢀ1.966
ꢀ1.880
ꢀ1.875
aminobutyronitrile
adiponitrile
76
24
16
2
glycolonitrile
4-aminobenzonitrile
2,2-dimethylcyclopropyl
cyanide
2-amino-2,3-
0
ꢀ1.691
dimethylbutanenitrile
benzonitrile
162
153
210
146
263
53
ꢀ2.244
ꢀ2.239
ꢀ2.576
ꢀ1.676
ꢀ2.800
ꢀ2.357
o-methoxyphenylacetonitrile
m-methoxyphenylacetonitrile
p-methoxyphenylacetonitrile
phenylacetonitrile
acrylonitrile
a Activity of nitrilase was assayed under the standard assay conditions
with 20 mM substrate. The activity with mandelonitrile was set as 100%.
were used as templates. A BLAST search of the Protein Data
Bank demonstrated the strongest similarity between the nitrilase
from A. faecalis ZJUTB10 and the protein PH0642 (PDB 1J31)/
PaNit (PDB 3IVZ) belonging to the nitrilase superfamily.
Comparative modeling was used to generate the most probable
structure of the query protein by the alignment with template
sequences, simultaneously satisfying spatial restraints and local
molecular geometry. Sequence identity between target and
templates were around 23% according to PDB, respectively.
Despite varying sequence identity, all nitrilase structures share a
characteristic monomer fold and contain glutamate, lysine, and
cysteine in their catalytic sites. The discrete optimized pro-
tein energy score in MODELER was also calculated to determine
the quality of protein structures. The generated structure was
The docking results (Table 4) indicated that nitrilase obtained
in this study could hydrolyze arylacetonitriles and aromatic
nitriles in the following preference: meta > ortho > para.
However, this nitrilase has no activity toward aliphatic nitriles
with long chains and nitriles with an amino group, which has
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Table 4. Substrates Docking Studies
substrate
molecular weight
docked energy (kcal/mol)
substrate
molecular weight
docked energy (kcal/mol)
benzonitrile
103.12
128.13
128.13
128.13
121.11
121.11
121.11
137.57
137.57
137.57
182.02
182.02
182.02
148.12
148.12
148.12
118.14
118.14
118.14
119.12
119.12
119.12
117.15
117.15
117.15
131.17
117.15
ꢀ2.244
ꢀ2.239
ꢀ2.538
ꢀ2.119
ꢀ1.568
ꢀ2.146
ꢀ2.151
ꢀ2.261
ꢀ2.564
ꢀ2.363
ꢀ2.320
ꢀ2.584
ꢀ2.122
ꢀ0.462
ꢀ1.593
ꢀ1.233
ꢀ1.630
ꢀ1.962
ꢀ1.880
ꢀ2.085
ꢀ2.420
ꢀ2.288
ꢀ2.223
ꢀ2.603
ꢀ2.423
ꢀ1.524
ꢀ2.800
mandelonitrile
o-methoxyphenylacetonitrile
m-methoxyphenylacetonitrile
p-methoxyphenylacetonitrile
p-fluorobenzylcyanide
p-nitrobenzylcyanide
p-aminobenzylcyanide
propionitrile
133.15
147.18
147.18
147.18
135.14
162.15
132.16
55.08
ꢀ2.664
ꢀ2.239
ꢀ2.576
ꢀ1.676
ꢀ1.696
ꢀ1.214
ꢀ1.601
ꢀ2.230
ꢀ2.357
ꢀ2.534
ꢀ2.652
ꢀ2.434
ꢀ2.665
ꢀ2.670
7.410
1,2-benzodinitrile
1,3-benzodinitrile
1,4-benzodinitrile
2-fluorobenzonitrile
3-fluorobenzonitrile
4-fluorobenzonitrile
2-chlorobenzonitrile
3-chlorobenzonitrile
4-chlorobenzonitrile
2-bromobenzonitrile
3-bromobenzonitrile
4-bromobenzonitrile
2-nitrobenzonitrile
3-nitrobenzonitrile
4-nitrobenzonitrile
2-aminobenzonitrile
3-aminobenzonitrile
4-aminobenzonitrile
2-hydroxybenzonitrile
3-hydroxybenzonitrile
4-hydroxybenzonitrile
o-tolunitrile
acrylonitrile
72.06
butyronitrile
69.11
isobutyronitrile
69.11
adiponitrile
108.14
94.12
glutaronitrile
succinonitrile
80.09
sebaconitrile
164.25
67.09
methacrylonitrile
aminoacetonitrile
aminobutyronitrile
glycolonitrile
ꢀ2.591
ꢀ1.896
ꢀ1.783
ꢀ1.966
ꢀ2.473
ꢀ2.492
ꢀ2.547
ꢀ2.515
ꢀ2.369
ꢀ2.548
ꢀ2.137
56.07
84.12
57.05
iminodiacetonitrile
2-furanocarbonitrile
2-pyridinacetonitrile
3-pyridinacetonitrile
2-cyanopyridine
95.10
93.08
209.25
209.25
104.11
104.11
104.11
m-tolunitrile
p-tolunitrile
3-cyanopyridine
3-phenylpropionitrile
phenylacetonitrile
4-cyanopyridine
been confirmed by the data listed in Table 3. The results of
docking also showed that this enzyme belongs to the arylaceto-
nitrilase family. Structure determination of the enzymeꢀsub-
strate complexes is needed to provide further evidence for this
conclusion.
and neutral pH favored its activity. Though several reports have
demonstrated that most nitrilases are ion-independent,27,28
A. faecalis ZJUTB10 nitrilase was enhanced by Mg2+; thus, the
relationship and function of Mg2+ with the nitrilase obtained in
this study need further investigation.
Modeling and mutation studies showed that conserved cata-
lytic residues Cys-163, Glu-47, and Lys-129 are the catalytic triad
of the nitrilase obtained in this study, and these residues are
essential in the activity of nitrilase. According to reports6,44 and
on the basis of the structural assignment, a section-conserved
sequence (residues 164ꢀ167 CEEH) was found in the substrate
binding pocket and is very close to the active site Cys163. The
‘enantioselective hot spot’ amino acids around 190 are situated in
the loop between α6 (α4 in the A. faecallis nitrilase) and β8.
These regions would be attractive targets for exploring higher
selectivity and activity of nitrilase in further studies using the
molecular modification of proteins.49
When a substrate molecule diffuses into the pocket, it can
bind onto the CEK of nitrilase in an orthogonal orientation
(Figure 9a). Glu-47 may further mediate the proton transfer from
the SH group of Cys-163, resulting in a strongly nucleophilic S
ion to initiate the first attack on the cyano carbon atom of the
substrate. In the CEK, cysteine attacks the cyano group of the
nitrile as a nucleophile, glutamate acts as a general base, and
lysine is involved in stabilization of the tetrahedral inter-
mediate.47 When (R)-mandelonitrile was docked in the active
site of nitrilase (Figure 9b), a hydrogen bond (bond length 3.5 Å)
was observed between the N (cyano nitrogen) of mandelonitrile
’ DISCUSSION
Nitrilases are important to many organisms because of their
ability to convert chemically reactive and harmful nitriles into
carboxylic acids as the sole product under mild conditions. As
promising biocatalysts, in recent years, nitrilases also have been
widely used in industrial applications for the preparation of
pharmaceuticals and other fine chemicals under mild conditions.
Because of the importance of nitrilases in industrial applications,
screening, and exploitation, development of new nitrilases has
recently received considerable attention. In this study, a new
A. faecalis nitrilase was obtained and cloned into E. coli cells.
Characterization of the recombinant enzyme showed that this
nitrilase has very excellent activity toward (R)-mandelonitrile
and other sets of nitriles. Especially, the nitrilase from A. faecalis
ZJUTB10 has a preference for arylacetonitriles. This study
further confirmed that none of the nitrilases show identical
properties. The most notable differences between the enzymes
are in substrate specificity, native structure and aggregation
properties, and pH optima.14
There are several factors that influence the function of
A. faecalis ZJUTB10 nitrilase. From this study, we found that this
nitrilase was more stable to heat compared to other nitrilases,38,43
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Journal of Agricultural and Food Chemistry
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group on (S)-mandelonitrile is 7.3 Å because its position is
hindered. This distance is beyond of the maximum catalytic
distance for this R-selective nitrilase (Figure 9c). Docked energies
for (R)-mandelonitrile (ꢀ2.664 kcal/mol) and (S)-mandelonitrile
(ꢀ2.013 kcal/mol) further confirmed that nitrilase in this study
can selectively catalyze (R)-mandelonitrile to (R)-mandelic acid.
Analysis of catalytic characteristics based on structure modeling
and molecular docking will provide new insight into nitrilase and
help to clarify the fundamentals for its application. To confirm our
molecular modeling and structural analysis, the crystal structure
of nitrilase should be determined, which is in progress in our
laboratory.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +86-571-88320614. Fax: +86-571-88320630. E-mail:
zhengyg@zjut.edu.cn.
Funding Sources
†The authors gratefully acknowledge the financial support of the
National High Technology Research and Development Program
of China (863 Program) (No. 2009AA02Z203), the Major Basic
Research Development Program of China (973 Project) (No.
2009CB724704), National Natural Science Foundation of China
(No. 31170761), Natural Science Foundation of Zhejiang Pro-
vince (No. Z4090612, Y4110409 and No. R3110155), and
Qianjiang Talent Project of Zhejiang Province.
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