Improvement of alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(-)-mandelic acid

Article abstract of DOI:10.1021/jf405683f

Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The K_m, V_max, and k_cat of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg^-1 min^-1, 24.45 s^-1, and 9.24 mM, 47.68 μmol mg ^-1 min^-1, and 34.55 s^-1, respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.

Full text of DOI:10.1021/jf405683f

Article
pubs.acs.org/JAFC
Improvement of Alcaligenes faecalis Nitrilase by Gene Site Saturation
Mutagenesis and Its Application in Stereospecific Biosynthesis of
(
R)‑(−)-Mandelic Acid
†
†
†
‡
,†
Zhi-Qiang Liu, Xin-Hong Zhang, Ya-Ping Xue, Ming Xu, and Yu-Guo Zheng*
†Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, People’s Republic of China
‡Zhejiang Laiyi Biotechnology Co., Ltd, Shengzhou, Zhejiang 312400, People’s Republic of China
*
S Supporting Information
ABSTRACT: Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of
carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene
site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After
mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on
product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/
Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis
nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for
6
h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid
after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was
2
3
1.50-fold higher than that of wild-type nitrilase. The K , V , and k of wild-type and Mut3 for mandelonitrile were 20.64 mM,
m max cat
3.74 μmol mg− min , 24.45 s , and 9.24 mM, 47.68 μmol mg min , and 34.55 s , respectively. A homology modeling and
1
−1
−1
−1
−1
−1
molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was
smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the
potential for industrial applications in the upscale production of (R)-(−)-mandelic acid.
KEYWORDS: nitrilase, molecular modification, gene site saturation mutagenesis, mandelonitrile, (R)-(−)-mandelic acid
INTRODUCTION
synthesis of (R)-(−)-mandelic acid has been widespread
■
because of its industrial development and application
Nitrilases (EC 3.5.5.1) are important industrial enzymes used
to convert nitriles directly into corresponding carboxylic acids
and ammonia in a single step with mild conditions.
Occurrences, catalytic mechanisms, applicabilities, and molec-
ular modifications of nitrilases have been widely reported.
By far, all known nitrilases can be classified as aromatic
1
1,12,15,16
prospects.
The production of (R)-(−)-mandelic acid
1
,2
by nitrilases has the most potential for the industrial
application.
1
1,15,17
There are several reported strains with
3
−5
abilities for the bioproduction of (R)-(−)-mandelic acid,
18
including Alcaligenes sp. ECU0401, Pseudomonas putida
7
,19
5,11
MTCC 5110
A. faecalis ZJUTB10,
A. faecalis ATCC
nitrilases, aliphatic nitrilases, and arylacetonitrilases according
20
8
21
6
8750, P. fluorescens EBC191, A. faecalis JM3, Bradyrhi-
zobium japonicum USDA110, and Burkholderia cenocepacia
J2315. However, the reported nitrilases still cannot meet the
requirement for the industrial production of (R)-(−)-mandelic
acid because of their poor activity and lower stability under low
pH. To resolve this problem, nitrilases from different origins
to substrate specificity. Nitrilases are found in many plants and
22
microorganisms, and their applications have been widely
23
6
explored. Recently, screening new nitrilases, exploring their
applications, and the molecular modification of nitrilases are
becoming hot issues because of the important application of
4
,5,7−10
nitrilases.
3
,5,24
have been modified by various approaches.
It was reported that one of the important industrial
applications of nitrilases is the conversion of (R,S)-mandeloni-
trile to (R)-(−)-mandelic acid. This reaction exhibits
excellent enantioselectivity, low-cost starting material, and
most especially the possibility of performing a dynamic kinetic
resolution of racemic substrate which provides theoretically
The gene site saturation mutagenesis method (GSSM) is
widely used in the modification of proteins based on the PCR
method, which has made a series of high-profile achievements
1
1
25
on nitrilases. Yeom et al. had mentioned that a positively
charged amino acid at position 129 in nitrilase from
Rhodococcus rhodochrous ATCC 33278 was an essential residue
for the activity with meta-substituted benzonitriles by the
1
1,12
1
00% yield of the product.
(R)-(−)-mandelic acid has been
used as an important intermediate for the production of
pharmaceuticals such as semisynthetic penicillins, cephalospor-
ins, antitumor agents, antiobesity agents, and chiral resolving
agent for the resolution of racemic alcohols and amines.
R)-(−)-mandelic acid can be produced by two routes
including chemical and biosynthetic methods. Biocatalytic
Received: December 24, 2013
Revised: March 29, 2014
Accepted: April 27, 2014
Published: April 28, 2014
1
2,13
(
14
©
2014 American Chemical Society
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2
6
GSSM. Wu et al. obtained a mutant nitrilase with increased
activity, specificity, and stability by modification at sites Thr210
and Leu201 located in α6, and Phe168 at the junction of the
TaKaRa company (Dalian, China). (R,S)-mandelonitrile, (R)-
(
−)-mandelic acid, and (S)-(−)-mandelic acid were purchased from
Sigma (St. Louis, MO). All other reagents and chemicals were
commercially available and of analytic grade.
2
7,28
β10 helix and α6.
cyanide hydratase and nitrilase activity in the protein were the
same by the GSSM. The cyanide dihydratase completely lost
its activity by the replacement of Cys163 to Ser, and the
replacement of Tyr53 to Phe caused an increase of the K value
of the enzyme for cyanide when the conserved amino acid
residues in the cyanide dihydratase from P. stutzeri AK61 were
altered by GSSM.
Nolan et al. found that the active sites for
Gene Site Saturation Mutagenesis. Random mutations were
introduced to the nitrilase gene by GSSM at positions of Leu175,
Ala197, Val305, Ala284, and Gln196 using PrimeSTAR HS DNA
polymerase (Takara) and pET28b-NIT plasmid as a template. The five
pairs of primers carrying mutations are listed in Table 1. The plasmids
with positive mutations from the previous round of mutations were
used as a template for the next step of mutation. The PCR reaction
mixture consisted of 200 μM for each dNTP, 0.2 μM primers, 10 μL of
29
m
30
2+
In this study, the recombinant nitrilase from A. faecalis was
selected to improve activity by using GSSM. On the basis of the
secondary and three-dimensional structures, interactions
5× PrimeSTAR Buffer (Mg plus), and 1.25 U PrimeSTAR HS DNA
polymerase (Takara) in 50 μL. The reaction was carried out at 94 °C
for 5 min, followed by 30 cycles of 98 °C for 10 s, 55 °C for 15 s for
amplifying the nitrilase gene, 72 °C for 8 min, and then 72 °C for 10
5
,31,32
between the substrate and nitrilase, and previous reports,
̈
min in a PCR thermocycler (Biometra, Gottingen, Germany). PCR
three sites in the α-helix (Leu175, Ala197, and Val305) and
one site in the β-sheet (Ala284) located after Cys163 were
selected for further mutagenesis. The site Gln196 was also
selected using the software HotSpot Wizard to identify the
point that plays an important role in enzyme catalysis and
substrate specificity. After mutagenesis, the mutants were
screened by a two-step screening method based on
bromocresol purple as the pH indicator combined with liquid
chromatography, and the best mutant Mut3 with serendip-
itously improved activity and pH stability was obtained. The
characteristics of purified wild-type and Mut3 were studied and
compared. The homology modeling and molecular docking
were further studied to elucidate the interactions between
substrate and nitrilase. Finally, the potential application in the
kinetic resolution of racemic mandelonitrile to prepare R-
products were subjected to 0.9% agarose electrophoresis and then
purified by a PCR purification kit (Simgen, Hangzhou, China). After
purification, the DNA fragment was digested with Dpn I at 37 °C for 3
h to remove the template plasmid containing the methylation sites.
The plasmid pET28b-NITmut was then transformed into E. coli BL21
3
3
(DE3) by the heat-shock method.
Screening Method. A two-step screening method which was
constructed to screen the mutants was described below: Each single
colony was picked from the plate with 50 mg/mL kanamycin (Kan) to
9
6-well plates with LB broth and incubated overnight with vigorous
shaking (500 rpm) at 37 °C. One part of the culture was transferred to
the new 96-well plates with 50 mg/mL Kan, 0.1 mM isopropyl-β-D-
thiogalactoside (IPTG), 100 mM (R,S)-mandelonitrile, and LB
medium, and then thermomixed for 8−10 h at 30 °C. (R,S)-
Mandelonitrile would be catalyzed to (R)-(−)-mandelic acid by
nitrilase expressed by IPTG induction. The final concentration of
(−)-mandelic acid was evaluated as well.
0
.01% (w/v) bromocresol purple solution was added for high-
throughput and rapid screening. The pH of the medium in the 96-well
plate was kept at 7.0, and the screening method was based on the
formation of yellow caused by mandelic acid as a result of hydrolysis
by positive mutants. When the pH became lower and lower with the
hydrolysis process of the (R,S)-mandelonitrile to produce mandelic
acid, the medium with purple background would change the color to
yellow. Finally, the positive mutants were selected by the formation of
yellow. At the same time, the mutants which made the color of
bromocresol purple solution change from purple to yellow were picked
from the corresponding plates and grown at 37 °C in 50 mL of LB
media containing 50 μL of 50 mg/mL Kan for further tests. The
nitrilase was induced by the addition of 0.1 mM IPTG when optical
density (OD) at 600 nm of the culture broth reached between 0.6 and
0.8. The cells were then incubated at 28 °C for 12 h and harvested by
centrifugation at 9000 rpm for 10 min. The reaction was carried out
with free cells in 1 mL of pure water at 40 °C, 500 rpm for 10 min.
After reaction, the reaction mixture was centrifuged at 12000 rpm for 5
min, and then the amount of (R)-(−)-mandelic acid in the supernatant
was subsequently determined by high-performance liquid chromatog-
raphy (HPLC) mentioned below to further confirm the positive
mutants with improved nitrilase activity.
MATERIALS AND METHODS
Strains, Plasmids, and Medium. E. coli BL21 (DE3) (F-ompT
gal dcm lon, hsdSB (rB- mB-) λ (DE3 (lacI lacUV5-T7 gene 1 ind1
■
Table 1. Primers for the Gene Site Saturation Mutagenesis
positions of
mutant
primers
Leu175
L175 (F):
′-CTCTGTACTCCGAANNNGCGCACGCTCTGTC-3′
5
L175 (R):
5′-GACAGAGCGTGCGCNNNTTCGGAGTACAGAG-3′
Ala197
Val305
Ala284
Gln196
A197 (F):
5
′-CTGTACTCCGAACAGNNNCACGCTCTGTC-3′
A197 (R):
5′-CAGAGCGTGNNNCTGTTCGGAGTACAGAGA-3′
V305 (F):
5′-GTCTAAATACGCCNNNTACTCCCAGCACGAAG-3′
V305 (R):
5′-CTTCGTGCTGGGAGTANNNGGCGTATTTAGAC-3′
A284 (F):
Nitrilase Purification and Sodium Dodecyl Dulfate−Poly-
acrylamide Gel Electrophoresis (SDS−PAGE). After being
induced by 0.1 mM IPTG, the cells harboring recombinant nitrilases
were collected by centrifugation and then were resuspended in 30 mL
of pure water and lysed by sonication with a Vibra-Cell VC 505
ultrasonic processor (Sonics and Materials Inc., Newtown, CT) at 400
W for 15 min. The resulting slurry was centrifuged at 12000 rpm for
5′-GAAGCCACTCGTCTGNNNCTGGATCTGGGTC-3′
A284 (R):
5′-GACCCAGATCCAGNNNCAGACGAGTGGCTTC-3′
Q196 (F):
5′-CATGGAAGAGATCNNNTTCGCTAAGGCTATC-3′
Q196 (R):
5′-GATAGCCTTAGCGAANNNGATCTCTTCCATG-3′
2
0 min, and the supernatant was loaded to a nickel-NTA superflow
column, which has been equilibrated with buffer (20 mM NaH PO
and 300 mM NaCl; and pH 8.0). Unbound proteins were washed out
from the column with buffer (20 mM NaH PO , 300 mM NaCl, 50
mM imidazole, and pH 8.0). The active enzyme was eluted with buffer
by a linear gradient from 50 to 500 mM imidazole at a flow rate of 1
mL/min. The purified enzymes were analyzed using 12% SDS−PAGE.
sam7 nin5))) (Invitrogen, Karlsruhe, Germany) was used for
heterologous expression. E. coli and recombinant E. coli strains were
grown in Luria−Bertani (LB) medium (tryptone 10 g/L, yeast extract
2
4
2
4
5
g/L, and NaCl 10 g/L). The pET28b-NIT plasmid and recombinant
5
nitrilase from A. faecalis was constructed and stored in our laboratory.
Dpn I and PrimeSTAR HS DNA polymerase were provided by the
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Enzyme Assays and Protein Assay. The standard activity assay
was carried out using (R,S)-mandelonitrile (100 mM) as substrate and
150 rpm in 200 mL reaction volume. The reaction mixture contained
100 mM potassium phosphate buffer (pH 8.0), 100 mM (R,S)-
mandelonitrile, and 5% wet cells of Mut3. (R,S)-mandelonitrile (100
mM) was fed in the subsequent 7 batches (total 800 mM) once an
hour. The reaction mixture (100 μL) was periodically withdrawn and
analyzed by HPLC.
0
.2 mg/mL of purified enzyme mixed in potassium phosphate buffer
(
100 mM and pH 8.0). The reaction mixture (total volume 10 mL)
was incubated at 45 °C for 10 min and stopped by HCl solution (final
2
2
M). Then, 200 μL of the mixture was centrifuged at 12000 rpm for
0 min, and the supernatant was subjected to HPLC analysis as
Isolation and Identification of (R)-(−)-Mandelic Acid. The
isolation procedure of (R)-(−)-mandelic acid was performed
according to the following procedure: 2% (w/v) activated carbon
was added into the fed-batch reaction mixture (200 mL) to remove the
cells. After filtration, the filtrate adjusted to pH 9.0−10.0 with 2 M
NaOH was extracted with an equal volume of ethyl acetate twice to
obtain the aqueous phase, which was acidified to pH 1.0−2.0 with 2 M
HCl. The aqueous phase suffered from vacuum filtration (SHB-III,
Zhengzhou trade Co., LTD, China) to remove the proteins in the
solvent, and then the filtrate was extracted with isopyknic ethyl acetate
three times. After the organic extract was dried by anhydrous sodium
sulfate, the solvent was removed through vacuum distillation
(Adyantag ML, Heidolph, Germany) in a rotary evaporator, and the
crystal of (R)-mandelic acid was subsequently obtained.
described below for assaying the nitrilase activity. The amount of (R)-
−)-mandelic acid was determined by a LC-20AD prominence liquid
(
chromatography 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
(
NH )H PO /CH OH = 13:7 (v/v, 1 mL/min) as the mobile phase,
4 2 4 3
and absorbance at 228 nm was measured. The retention time for (R)-
(
−)-mandelic acid was 3.2 min. The optical purity of (R)-
(−)-mandelic acid was determined by an 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
The 13C and H nuclear magnetic resonance (NMR) spectra of (R)-
(−)-mandelic acid were obtained on a NMR spectrometer (AVANCE
500 MHz, Bruker, Fallanden, Switzerland) with DMSO as the solvent,
using 500 and 125 MHz for carbon and proton determinations,
respectively. The FTIR spectrum for (R)-(−)-mandelic acid was
recorded on a Fourier transform infrared spectrometer (Nicolet 6700;
Thermo, Waltham, MA) with a Contimuμm FTIR Micro system in
1
(
90:10:0.1, v/v) at a wavelength of 228 nm.
One unit of nitrilase activity was defined as the amount of enzyme
that produces 1 μmol of (R)-(−)-mandelic acid per minute under the
conditions described above. Protein quantitative analysis was
34
conducted using the Bradford method with bovine serum albumin
as a standard.
Optimum pH and pH Stability. To investigate the optimal pH
and the effect of pH on the activity of nitrilase, four buffers including
citrate buffer (pH 3.0−6.0), phosphate buffer (pH 6.0−8.5), Tris-HCl
buffer (pH 7.0−8.5), and glycine-NaOH buffer (pH 8.5−10.0) with a
concentration of 100 mM were used, and the reactions were
performed at 45 °C using 100 mM (R,S)-mandelonitrile as substrate.
To investigate pH stability, the enzyme was dissolved in buffers of pHs
ranging from 6.5 to 8.5 and incubated at 0 °C from 0.5 to 6 h, and
then the relative activities were performed with standard assay
conditions. Activity without treatment obtained at pH 8.0 was used as
a blank and regarded as 100%.
−1
the region of 4000−400 cm using KBr pellets. The LC-MS spectrum
for (R)-mandelic acid was recorded on a high performance liquid
chromatography−time-of-flight mass spectrometer (Agilent Technol-
ogies 1200 Series, 6210 Time-of-Flight MS spectrometer) (Agilent
Technologies, Waldbronn, Germany), and the experimental parame-
ters were used as follows: ionization mode, ESI scan; gas temperature,
3
00 °C; fragmentor, 80 V; drying gas, 3.5 L/min; skimmer, 65 V;
nebulizer, 20 psig; OCTIRFV pp, 250 V; and Vcap, 3000 V.
Statistical Analysis. If not specifically noted, all experiments in
this study were performed in triplicate. Analysis of variance (ANOVA)
was carried out using the SAS program, version 8.1 (SAS Institute Inc.,
Cary, NC). Least significant differences (LSD) were computed at P <
Optimum Temperature and Thermal Stability. The optimal
temperature of the enzyme was determined by assaying enzyme
activities at temperatures ranging from 25 to 65 °C. Thermal stability
was investigated by incubating enzymes at temperatures ranging from
0
.05.
3
0 to 60 °C. The enzyme was incubated in a heat-block at the specified
RESULTS
■
temperature for a certain time. The relative activities were examined at
Construction and Screening of Mutant Libraries. The
PCR products of GSSM were purified and digested by Dpn I to
4
5 °C for 10 min.
Circular Dichroism (CD) Measurements. CD spectra were
recorded on a JASCO J-815 Spectropolarimeter (JASCO Inc., Easton,
MD) with Spectra Manager 228 software. The temperature was
controlled from 10 to 60 °C 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 0.02 mg/mL
nitrilase was generated in ddH O. The spectra were recorded from 190
2
to 250 nm.
Homology Modeling and Molecular Docking. The three-
dimensional homology model of nitrilase was constructed using Build
Homology Models (MODELER) in Discovery studio (DS) 2.1
(
Accelrys Software, San Diego) and the crystal structure of a
hypothetical protein PH0642 (PDB accession code 1J31) was used
as the template. The catalytic tunnel size was analyzed using software
HotSpot Wizard to identify the residues essential for protein function,
enzyme activity, and substrate specificity. Autodock 4.0 was used to
study the interaction between substrate and nitrilase, and to predict
the spatial structure and catalytic mechanism of the nitrilase.
Figure 1. Mutant library screening for the high activity of nitrilases in a
96-well plate.
Time Course of (R,S)-Mandelonitrile Hydrolysis. The reaction
mixture (10 mL) contained 0.1 g of wet cells of wild-type or evolved
nitrilase suspended in 100 mM potassium phosphate buffer (pH 8.0)
containing 100 mM (R,S)-mandelonitrile. These reactions were carried
out at 40 °C in a reciprocal shaker at 150 rpm. Samples were removed
at predetermined times, and the yield was determined by HPLC.
Fed-Batch Production of (R)-(−)-Mandelic Acid Using Mut3
as the Catalyst. The fed-batch reaction was performed at 40 °C and
remove the template plasmid containing the methylation sites
and to create the recombination library of pET28b-NITmut
.
Single colonies of positive mutants were picked from LB agar
plates containing Kan resistance to 96-well plates with 200 μL
of LB broth supplemented with 50 mg/mL of Kan and
incubated overnight with vigorous shaking (500 rpm) at 37 °C.
Ten microliters of the culture was transferred to the new 96-
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Figure 2. SDS−PAGE analysis of purified nitrilases. Gels with 30%
polyacrylamide were subjected to protein analysis, and the protein
bands were stained with Coomassie blue R250. Lanes 1 and 2 stand for
Mut3 and the wild-type nitrilase, respectively.
well plates with 50 mg/mL Kan and 500 μL of LB medium, and
then was incubated at 37 °C until the OD at 600 nm of the
culture broth reached between 0.6 and 0.8 for retesting the
activity and pH stability. In addition, plates containing both 100
mM (R,S)-mandelonitrile and 0.1 mM IPTG were incubated
for 10 h at 28 °C. The final concentration of 0.01% (w/v)
bromocresol purple solution was added for screening. The wells
containing positive colonies with improved properties could
become yellow and the negative mutants remain in the purple
background (Figure 1 and Figure S1 in the Supporting
Information). Over 6300 colonies in total were screened, and
5
5 clones could make the wells turn yellow. Among these
Figure 3. Influence of pH on the activity of nitrilases and stability. (A)
Reactions were performed at 45 °C with different buffers. Hollow and
solid represent the mutant Mut3 and the wild-type nitrilase,
respectively. (B) pH stability was investigated by dissolving the wild-
type nitrilase in phosphate buffers of pHs ranging from 6.5 to 8.5 and
incubated at 0 °C from 0.5 to 6 h. (C) pH stability was investigated by
dissolving Mut3 in phosphate buffers of pHs ranging from 6.5 to 8.5
and incubated at 0 °C from 0.5 to 6 h.
clones, 10 better ones were retested using HPLC, and the clone
with the greatest improvement in activity was selected (Table
S1 in the Supporting Information). At the same time, selected
variants were sequenced, and their amino acid substitutions are
listed in Table S1 (Supporting Information). After three rounds
of GSSM followed by screening, a best mutant Mut3
(
Gln196Ser/Ala284Ile), with 200% relative activity than the
wild-type, was selected as the targeted evolved nitrilase for
further studies.
Expression and Purification of Nitrilases. The wild-type
and evolved nitrilases were subjected to Nickel-NTA affinity
chromatography. As shown in SDS−PAGE (Figure 2), there is
only one pure band with a molecular weight of around 43 kDa
observed for both of the purified wild-type and evolved
nitrilases, which is consistent with the predicted molecular
weight.
Influences of pH Values on the Activites of Nitrilases.
It is well-known that the pH values affect the ionic environment
of the enzyme, thus acting on its interaction between substrate
and enzyme activity. To investigate the effect of pH on nitrilase
activity, the reaction was performed in buffers including citrate
buffer (pH 3.0−6.0), phosphate buffer (pH 6.0−8.5), Tris-HCl
buffer (pH 7.0−8.5), and glycine-NaOH buffer (pH 8.5−10.0)
with a concentration of 100 mM at 45 °C. As shown in Figure
3A, both wild-type nitrilase and Mut3 showed almost the same
trends of activities under different pH conditions. The
maximum specific activity of the evolved and wild-type
nitrilases was observed at pH 8.0. Compared to wild-type
nitrilase, the activity of Mut3 has increased significantly ranging
from pH 3.0 to 10.0. Moreover, at pH 8.0, the specific activity
of Mut3 (42.77 U/mg) was improved 154% compared to that
of wild-type nitrilase (27.79 U/mg).
The pH stability study (Figure 3B and C) showed that Mut3
was more stable at pH 6.5 with about 90% residual activity after
6 h than the wild-type nitrilase with only 76% residual activity.
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Figure 5. spectroscopic structural analyses of nitrilases. CD wave-
length scans of the wild-type nitrilase (A) and Mut3 (B) at pH 7.5.
Figure 4. Effect of temperature on the activity of nitrilases and
stability. (A) Reactions were performed in 100 mM at pH 8.0
phosphate buffer at various temperatures ranging from 25 to 65 °C.
(
B) Thermal stability was investigated by incubating the wild-type
nitrilase at temperatures ranging from 30 to 60 °C. (C) Thermal
stability was investigated by incubating Mut3 at temperatures ranging
from 30 to 60 °C.
Effects of Temperatures on the Activities. The effects of
temperatures on nitrilase activities of the wild-type and Mut3
were investigated. Enzyme (0.2 mg/mL) was dissolved in 100
mM phosphate buffer (pH 8.0) and incubated for 5 min at a
temperature ranging from 25 to 65 °C before reaction. The
reaction was started by adding 100 mM (R,S)-mandelonitrile
under the corresponding temperature at 150 rpm for 10 min in
a reciprocal shaker. As shown in Figure 4A, the optimum
temperatures were 45 °C for both the wild-type and Mut3. The
activities of nitrilases decreased significantly when the temper-
ature was above 45 °C, and the specific activity of Mut3 was
about 154% higher as compared to the wild-type nitrilase under
the optimal temperature and pH.
Figure 6. Reaction rate at different mandelonitrile concentrations.
Reactions were performed at 45 °C, 150 rpm, with 100 mM potassium
phosphate buffer (pH 8.0).
the residual activity of Mut3 was 75% at 30 °C. After incubation
for 1 h at 50 and 60 °C, the relative activity of wild-type
nitrilase was sharply reduced to 4.40% and 1.03%, and Mut3
was 6.30% and 1.92%, respectively. Mut3 was unstable at higher
temperature as revealed by a half-life of 54 h at 30 °C, 1.9 h at
40 °C, and 0.4 h at 50 °C, which was better than that of the
wild-type nitrilase with half-lives of 21 h at 30 °C, 1.8 h at 40
°C, and 0.2 h at 50 °C (Figure 4B and C). The results
corresponded to the circular dichroism data, indicating the
The study of thermostability showed that the activity of wild-
type nitrilase maintained about 60% incubation after 16 h, while
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Figure 7. Time course analysis of (R,S)-mandelonitrile biotransforma-
tion by wild-type nitrilase and Mut3 under optimal conditions with a
pH of 8.0, a temperature of 40 °C, and a concentration of (R,S)-
mandelonitrile of 100 mM.
Figure 9. Tunnel analyses of wild-type nitrilase (A) and Mut3 (B).
calculated according to the Lineweaver−Burk plots. The values
of K , V , and k were calculated to be 20.64 mM, 33.74
m
max
cat
−
1
−1
−1
μmol mg min , and 24.45 s for wild-type nitrilase, while
they were 9.24 mM, 47.68 μmol mg min , and 34.55 s for
Mut3, respectively.
−1
−1
−1
Time Course of (R,S)-Mandelonitrile Hydrolysis. Wet
cells of wild-type or Mut3 (0.1 g) was suspended in the 10 mL
reaction mixture containing 100 mM potassium phosphate
buffer (pH 8.0) and 100 mM (R,S)-mandelonitrile. The
reactions were carried out at 40 °C in a reciprocal shaker at
150 rpm. Samples were removed at predetermined times, and
the yield was determined by HPLC analysis. As shown in
Figure 7, the yield of Mut3 could reach 100% in 0.5 h which
was higher than that of the wild-type, and the enantiomeric
excess (e.e.) of (R)-(−)-mandelic acid was >99%.
Fed-Batch Production of (R)-(−)-Mandelic Acid. To
obtain efficient production of (R)-(−)-mandelic acid and
reduce the potential inhibition on enzyme at high substrate
concentration, a fed-batch reaction was performed using Mut3
as catalyst. (R,S)-Mandelonitrile (100 mM) was fed to the
subsequent 7 batches (total concentration of substrate: 800
mM) once an hour. The accumulative concentration of (R)-
Figure 8. Homology modeling and docking analysis of nitrilases with
mandelonitrile docking into the active site of the wild-type nitrilase
(
A) and Mut3 (B).
nitrilases were sensitive to heat (Figure 5 and Table S2,
Supporting Information).
Kinetic Analysis of Wild-Type and Evolved Nitrilase.
To determine the kinetics of reaction catalyzed by nitrilases, the
initial rate V was investigated at different concentrations (0−
0
100 mM) of (R,S)-mandelonitrile (Figure 6). The experimental
data were fitted to the Michaelis−Menten equation (V = (V
0
max
[
S])/(K + [S])), and the values of K and V
were
max
m
m
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Figure 10. Identification of (R)-(−)-mandelic acid. LC-MS spectrum of (R)-(−)-mandelic acid, standard (R)-(−)-mandelic acid (A), and separated
R)-(−)-mandelic acid (B).
(
(
−)-mandelic acid reached 585 mM after 5.5 h of
with the mass of (R)-(−)-mandelic acid. The FTIR spectrum of
the separated (R)-(−)-mandelic acid is presented in Figure 11A
and B; the two sharp peaks presented at 3448 and 1724 cm−
could be assigned to the O−H and CO stretching vibrations,
respectively. The two peak absorption bands at 1495 and 1455
biotransformation (Figure S2, Supporting Information).
3
5
1
Compared to the previous report, 693 mM of (R)-
−)-mandelic acid concentration at 7.5 h in this work displayed
(
the highest productivity in terms of the nitrilase catalyzing
asymmetric hydrolysis of (R,S)-mandelonitrile, and the optical
purity of the (R)-(−)-mandelic acid was maintained above 99%
during the reaction process. In addition, considering the cost of
industrial application, Mut3 with excellent tolerance against
high product concentration is more favorable for applications
because the separation cost of product will be reduced
substantially. Taking the volumetric productivity and product
concentration tolerance into account, the mutant Mut3 was
potential for industrial application.
−1
cm were due to the CC stretching vibration on the phenyl
13
group. When C NMR was performed, the isolated product
gave one signal at 174 ppm, which referred to the C positions
of the carboxyl group. The absorption bands between 126 and
140 ppm were due to the C positions on the phenyl group
(Figure S4, A and B, Supporting Information). Figure S5, A and
B (Supporting Information) shows four different kinds of H
atoms, which corresponded to the structure of (R)-(−)-man-
delic acid, one peak at chemical shift δ = 12.6356 ppm
representing one H atom at the carboxyl group. The split peaks
at chemical shifts between δ = 7.2797 ppm and δ = 7.4522 ppm
represented the H positions on the phenyl group, and the peak
at chemical shift δ = 2.50 ppm referred to the H position of the
hydroxyl group. Accordingly, it was concluded that the isolated
product was (R)-(−)-mandelic acid.
Isolation and Identification of (R)-Mandelic Acid. After
isolation and purification of (R)-(−)-mandelic acid according
to the method described in Materials and Methods, (R)-
(−)-mandelic acid with 99% purity was obtained (Figure S3,
Supporting Information). The LC-MS spectrum of the
separated (R)-(−)-mandelic acid was presented in Figure 10A
and B, LC-MS showed that the molecular ion peak of (R)-
(−)-mandelic acid was m/z 151.0, which was in accordance
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Figure 11. Identification of (R)-(−)-mandelic acid. Infrared spectroscopy of (R)-(−)-mandelic acid, standard (R)-(−)-mandelic acid (A), and
separated (R)-mandelic acid (B).
Table 2. Comparison of Wild-Type Nitrilase and Mut3 with Reported Nitrilases
substrate (mM)
bacteria
resting cells (g)
reaction time (h)
the volume of reaction (mL)
yield (%)
reference
100
100
100
100
E. coli JM109/pNLE
E. coli M15/BCJ2315
the wild-type nitrilase
Mut3
0.01
0.1
2
1
100
99.1
100
100
15
1
10
10
10
37
0.1
1
this work
this work
0.1
0.5
5
,11,12
DISCUSSION
lectivity, low cost, and almost 100% yield.
The goal of this
■
study is to improve the nitrilase from A. faecalis to meet the
requirement of an upscale production of (R)-(−)-mandelic
acid.
Nitrilases have recently drawn considerable attention due to
their ability to transform nitriles to carboxylic acids with
3
6,37
chemo-, region-, and enantioselectivity.
important industrial enzymes, have more significant improve-
ments than traditional chemical catalysts, which have
mediated the industrial conversion of aliphatic, arylaliphatic,
aromatic, and heterocyclic nitriles.
industrial applications of nitrilases is the conversion of (R,S)-
mandelonitrile to (R)-(−)-mandelic acid with high enantiose-
Nitrilases, as
On the basis of the secondary structure, relationships
5,31,32
between substrate and the nitrilase, and previous reports,
3
8
five sites (Leu175, Ala197, Val305, Ala284, and Gln196) were
selected to carry out mutations for improving activity of
nitrilase. After screening by a two step screening method, the
best mutant Mut3 was selected. Mut3 displayed dramatic
improvement in activity, pH stability, and tolerance to the
6,37
One of the important
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substrate. In a fed-batch reaction with 800 mM (R,S)-
mandelonitrile as the substrate, the cumulative concentration
of (R)-(−)-mandelic acid in the reaction mixture after 5.5 h of
biotransformation reached 585 mM with an e.e. of 99%, which
was higher than that in a previous report with 520 mM after 18
h. At the same time, the space-time productivity of Mut3 (10.64
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: +86-571-88320630. Fax: +86-571-88320630. E-mail:
zhengyg@zjut.edu.cn.
Funding
mmol L−1
−1
−1
This work was financially supported by the National High
Technology Research and Development Program of China
863 Program) (No. 2011AA02A210), Natural Science
Foundation of Zhejiang Province (No. R3110155, Z4090612
and Y4110409), and Qianjiang Talent Project of Zhejiang
Province.
h
g ) was 7.40-fold higher than that in a previous
−1
−1 −1 35
report (1.44 mmol L
h
g ).
(
CD results confirmed that these nitrilases are sensitive to
heat and that the thermostability of Mut3 was better than that
of wild-type nitrilase. The nitrilase activites of both wild-type
and Mut3 shared almost the same trends under different pH
conditions. The specific activity of Mut3 (42.77 U/mg) was
Notes
The authors declare no competing financial interest.
154% higher than that of wild-type nitrilase (27.79 U/mg) at
pH 8.0. The analysis of the time course of (R,S)-mandelonitrile
hydrolysis indicated that the yield of Mut3 could reach 100% in
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