Improving the catalytic efficiency and stereoselectivity of a nitrilase from: Synechocystis sp. PCC6803 by semi-rational engineering en route to chiral γ-amino acids

Article abstract of DOI:10.1039/c8cy02455c

Nitrilase-catalysed desymmetrization of 3-substituted glutaronitriles to optically active 3-substituted 4-cyanobutanoic acid offers an attractive approach to access chiral β-substituted γ-amino acids, an important moiety in pharmaceuticals. In this study,

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DOI: 10.1039/C8CY02455C
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Improving catalytic efficiency and stereoselectivity of a nitrilase
from Synechocystis sp. PCC6803 by semi-rational engineering en
route to chiral -amino acids
Received 00th January 20xx,
Accepted 00th January 20xx
Shanshan Yu,a† Peiyuan Yao,a† Jinlong Li,a Jinhui Feng,a Qiaqing Wu,a* and Dunming Zhua*
DOI: 10.1039/x0xx00000x
Nitrilase-catalysed desymmetrization of 3-substituted glutaronitriles to optically active 3-substituted 4-cyanobutanoic acid
offers an attractive approach to access chiral -substituted -amino acids, an important moiety in pharmaceuticals. In this
study, we employed enzyme-substrate docking and alanine scanning to determine the key amino acid residues which had
a positive effect on the activity and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 (SsNIT). Then site-
saturation mutagenesis and combinatorial mutagenesis of the positive amino acid residues (H141, P194, M197, I201 and
F202) were performed, and double mutant (P194A/F202V) and triple mutant (P194A/I201A/F202V) with high activity and
stereoselectivity were identified for desymmetrization of 3-substituted glutaronitriles to afford (S)-3-substituted-4-
cyanobutanoic acids with high space-time productivity of up to 488 g·L-1·d-1. The molecular calculation suggests that the
enzymatic activity improvement may be due to the enlargement of the substrate binding cavity. It is interesting that the
stereoselectivity is enhanced simultaneously.
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Synechocystis sp. strain PCC 6803 could catalyse the
desymmetrization of 3-substituted glutaronitriles to produce
optically active 3-substituted 4-cyanobutanoic acid, which could
be transformed to (S)-Pregabalin and (R)-Baclofen by Curtius
rearrangement and hydrolysis.33 However, the catalytic
efficiency of these nitrilases towards 3-substituted
glutaronitriles does not meet industrial requirements.
Introduction
Chiral -amino acids have been attracted increasing attention
for their biological activity and pharmaceutical importance.1-3
They are key building blocks for the marketed drugs such as
Pregabalin (Lyrica®) which was used to treat epilepsy,
neuropathic pain, fibromyalgia and generalized anxiety
disorder,4, 5 the muscle relaxant and antispastic agent Baclofen
(Lioresal®),6, 7 and its prodrug Arbaclofen placarbil which can
enhance oral absorption compared to Baclofen.8, 9 In addition,
their ring-closed products, -lactams, are also widely applied in
clinic therapeutics, such as the antidepressant drug (R)-
Rolipram,10 and antiepilepsy drug Brivaracetam (Briviact®)
(Figure 1).11-13 Numerous chemical methods have thus been
developed for the preparation of these compounds.14-21
Recently, biocatalysis has become an increasingly attractive
approach to prepare chiral pharmaceutical intermediates
because of its environmentally benign, excellent selectivity and
low cost,22-25 and a large number of methods involving lipase,26,
27
33
transaminase,28 ene-reductase,29-31 nitrilase32,
and 4-
Figure 1. Selected biologically active compounds with chiral -
amino acids or -lactam.
oxalocrotonate tautomerase34 have been established en route
to chiral -amino acids. In our previous work, we have found
that nitrilases HsNIT from Herbaspirillum sp. GW103, BjNIT6402
from Bradyrhizobium japonicum USDA110, and SsNIT from
Directed evolution is a versatile tool for manipulating the
catalytic property of enzymes, including stereoselectivity,
activity and substrate scope.35-42 In this study, we attempted to
engineer SsNIT to improve its activity and stereoselectivity
towards 3-substituted glutaronitriles. Since the crystal structure
of SsNIT (PDB ID: 3WUY) has been reported,43 3-(4-
chlorophenyl) glutaronitrile (1a) was docked into the active site.
The key residues lining the substrate binding pocket were
investigated by alanine scanning. Further site-saturation
a. National Engineering Laboratory for Industrial Enzymes, Tianjin Engineering
Research Center of Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport
Economic Area, Tianjin 300308, People’s Republic of China. Email:
zhu_dm@tib.cas.cn; wu_qq@tib.cas.cn
† These authors contributed equally.
Electronic Supplementary Information (ESI) available: See DOI: 10.1039/x0xx00000x
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mutagenesis and iterative combinatorial mutagenesis P194V, I201R and I201L) improved the catalytic activity in the range
View Article Online
DOI: 10.1039/C8CY02455C
generated variants with higher activity and stereoselectivity, of 1.2 to 2.2-fold relative to wt SsNIT.
which were applied in the preparation of a variety of chiral 3-
substituted 4-cyanobutanoic acids.
Table 1 Relative activity and stereoselectivity of mutant
enzymes towards 1a by using resting cells
relative activitya
(%)
ee
(%)
Results and discussion
Alanine scanning
Enzymes
WT
100
NDb
34
97(S)
ND
In our previous work, SsNIT could catalyse hydrolysis of 3-(4-
chlorophenyl) glutaronitrile (1a) to afford (S)-3-(4-
chlorophenyl)-4-cyanobutanoic acid ((S)-2a).33 To further
improve its activity and stereoselectivity towards 3-substituted
glutaronitriles, especially 1a, we docked 1a into the binding
pocket of SsNIT (PDB ID: 3WUY) using Discovery Studio 4.1
(Figure S1 in the Supporting Information). Fourteen amino acid
residues located within a distance of 5Å of 1a were chosen and
mutated to alanine. The relative activity, by-product amide and
enantioselectivity of the variants were analysed by chiral HPLC.
No amide was detected for all the mutants. As shown in Table
1, the activities of five mutants (H141A, P194A, M197A, I201A
and P202A) toward 1a were significantly improved relative to
that of wt SsNIT, and no tradeoff occurred in stereoselectivity.
Whereas other five mutants (Y59A, T139A, E142A, W170A and
F193A) had almost no activity, the mutants (F64A, Y140A,
V198A and Q205A) showed lower activity compared to wt
SsNIT. Interestingly, the ee value of mutant V198A was 75% (S),
indicating that residue V198 might have a dramatic effect on the
stereoselectivity towards 1a. Thus the residues H141, P194,
M197, I201 and F202, which showed positive effect on the
activity and stereoselectivity towards 1a, were selected for
further studies.
Y59A
F64A
>99(S)
ND
T139A
Y140A
H141A
E142A
W170A
F193A
P194A
M197A
V198A
I201A
F202A
Q205A
ND
82
>99(S)
90(S)
ND
158
ND
ND
ND
338
129
71
ND
ND
>99(S)
>99(S)
75.2(S)
>99(S)
>99(S)
>99(S)
204
541
68
Site-saturation mutagenesis and combinatorial mutagenesis
The site-directed mutagenesis libraries of five positive residues
(H141, P194, M197, I201 and F202) were constructed by using the
reduced amino acid alphabet (NDT). The mutant libraries were
screened by phenol hypochlorite colorimetry method, a process that
NH3 reacted with hypochlorite-alkaline phenol to form blue
a The activity of wild type SsNIT (WT) towards 1a was defined as
indophenol, which could be quantitatively detected by using a 100%. b ND means not detected.
44, 45
spectrometer at 630 nm to acquire the NH3 concentration.
Highly active variants towards 1a in the five libraries were rapidly
In order to further improve the catalytic efficiency of SsNIT
identified in 96-well plates containing wt SsNIT as control. The towards 1a, combinatorial mutagenesis was performed on the best
relative activity, generation of by-product and enantioselectivity of mutant F202V, because beneficial mutations often have an additive
the hits were determined by chiral HPLC analysis. As shown in Table effect.46 Consequently, three double mutants H141D/F202V,
2, the residue F202 had a significant effect on the activity of SsNIT P194A/F202V and I201A/F202V were constructed and assayed
towards 1a. When F202 was mutated to smaller amino acids, such as towards 1a. As shown in Table 2, mutant P194A/F202V showed the
F202A, F202I, F202V and F202S, they were observed higher activity. highest activity towards 1a (14.6-fold relative to wt SsNIT), while
Especially F202V showed 6.25-fold improvement in activity towards H141D/F202V was inactivated unexpectedly. Thus mutant
1a relative to that of wt SsNIT. We surmise that the bulky side chain P194A/F202V was selected as a template for the last round of
of F202 at the entrance of the binding pocket may be involved in a mutagenesis, which resulted in a triple mutant P194A/I201A/F202V.
steric clash with the unreactive cyano group of 1a, preventing the The variant P194A/I201A/F202V exhibited the highest catalytic
other cyano group moving into the catalytic triad. The residue M197 activity (20.8-fold relative to wt SsNIT) and excellent
located at the middle of
a long substrate-binding loop is enantioselectivity with formation of only a little by-product amide
an important part of the big binding pocket, which has a great effect (1%).
on the stereoselectivity or amide formation. Thus variant M197V
showed lower stereoselectivity (92% ee) and variant M197C Table 2 Relative activity, stereoselectivity and amide formation of
produced more byproduct amide (14%). Other mutants (H141D, mutant enzymes towards 1a by using resting cells
2 | J. Name., 2012, 00, 1-3
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Relative activitya
(%)
ee
(%)
Amide
(%)
entrance of the substrate-binding cavities. The volumes increased
View Article Online
DOI: 10.10399/C983CYÅ03245o5fC
Enzymes
almost 100 Å3 from 897 Å3 of wt SsNIT to
P194A/I201A/F202V. The three position are thus supposed to
affect the steric effects.
WT
100
219
134
79
97(S)
>99(S)
>99(S)
92(S)
NDb
ND
ND
ND
14
H141D
P194V
M197C
M197V
I201L
Table 3 Enzymatic reaction kinetics of SsNIT and its mutants for 1a
a
Km
Vmax
kcat
kcat/Km
Enzymes
WT
(mM) (μM·min-1·mg-1) (s-1) (mM-1·s-1)
0.25±
0.43±
170
106
122
205
90
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
0.32±0.04
0.28±0.01
1.69
0.97
5.96
0.07
0.05
ND
ND
ND
ND
ND
ND
0.38±
0.37±
I201R
F202I
P194A
F202V
0.02
0.01
0.13±
0.55±0.1 0.7±0.1
F202G
F202S
F202V
0.09
258
625
0.31±
3.48±
2.60±0.13
P194A/F202V
11.02
11.17
0.06
0.15
F202Y
140
ND
>99(S)
ND
ND
4.89±
0.51
P194A/I201A/F202V 0.44±0.1 3.66±0.39
H141D/F202V
ND
a The value of kcat was calculated with dimeric molecular weight
because the dimer was suggested as functional unit for nitrilase
catalysis43.
P194A/F202V
I201A/F202A
1461
368
>99(S)
>99(S)
99(S)
ND
ND
1
P194A/I201A/F202V
2085
a The activity of wild type SsNIT (WT) towards 1a was defined as
100%. b ND means not detected.
Enzymatic kinetics
Wild type SsNIT and its mutants P194A, F202V, P194A/F202V and
P194A/I201A/F202V were purified by Ni2+ affinity column. The
denaturation midpoint temperatures of the purified variant
enzymes were determined by Ultrasensitive differential scanning
calorimeter (NanoDSC), and they were 5 - 10C lower than that of
wild-type enzyme (58C). The kinetic parameters of purified
mutant enzymes in Table 3 were determined by HPLC analysis and
calculated through nonlinear curve fit by Origin 8.0. It was
observed that Km and kcat/Km of F202V towards 1a were 0.13 mM
and 5.96 mM-1·s-1, while those of wt SsNIT were 0.25 mM and 1.69
mM-1·s-1,
respectively.
Mutants
P194A/F202V
and
P194A/I201A/F202V had the highest catalytic efficiency (over 6-
fold relative to wt SsNIT). But P194A/I201A/F202V showed the
highest reaction rate 3.66 μM·min-1·mg-1 (11.4-fold relative to
that of wt SsNIT), which was 2.6 μM·min-1·mg-1 for P194A/F202V
(8.2-fold relative to that of wt SsNIT). The residue P194 was near
the catalytic triad E53 and C169. When it was mutated to small
amino acids, it is easier for the reactive cyano group of 1a to move
Figure 2 Illustration of the substrate-binding cavities of wt SsNIT
(green) and its mutant P194A/I201A/F202V (red). Active pocket
volumes of wt SsNIT and its mutant P194A/I201A/F202V are 897Å3
and 993 Å3.
Analytical scale enzymatic desymmetrization of 3-substituted
glutaronitriles
To explore the substrate spectrum of wt SsNIT and its mutant
P194A/I201A/F202V, a series of 3-substituted glutaronitriles 1a-n
were screened by phenol hypochlorite colorimetry and chiral HPLC
close to the catalytic triad. The active site volumes were
48
calculated by POVME (POcket Volume MEasurer)47,
and
exhibited by PyMOL (The PyMOL Molecular Graphics System:
Version 2.0 Schrödinger, LLC) in Figure 2. It is rather obvious that
the spaces are enlarged both in the active center and at the
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analysis to determine the specific activity and ee values, respectively. up to 99% for variant P194A/I201A/F202V. VAiefwteArrticlseiOmnplinlee
As shown in Table 4, the specific activity of P194A/I201A/F202V was centrifugation, acidification, extraction andDrOemI: 1o0v.a10l 3o9f/Cth8eCYs0o2lv4e5n5Ct,
2.30 U/mg with 99% ee value, which was 11-fold relative to that of the desired product (S)-2a was obtained as white solid in 91% yield
wt SsNIT (0.21U/mg) with 97% ee value toward 1a. There was almost and 99% ee values. The space-time productivity of mutant
no difference between wt SsNIT and the mutant on substrate bias, P194A/I201A/F202V is 488 g·L-1·d-1, which is 34.4 fold relative to that
and their activity was primarily dependent on steric effect. of nitrilase HsNIT from Herbaspirillum sp. GW103 (18.5 g·L-1·d-1). 33
Compared to other substrates, the catalytic efficiency of
P194A/I201A/F202V toward 1n showed more significant changes
which was 78-fold relative to wt SsNIT. Due to enlargement of the
substrate-binding pocket, variant P194A/I201A/F202V displayed
higher specificity activity toward those bulky substrates. It is
interesting that the stereoselectivity was also enhanced. For
instance, the activity toward substrate 1h was 5-fold relative to that
of wt SsNIT and the ee value increased from 74% (S) to 97% (S).
Table 4 Substrate spectrum of SsNIT and its triple mutant
WT
P194A/I201A/F202V
specific
activitya
(U/mg)
specific
eeb
Substrate (R)
Enhancement
fold
eeb
(%)
activitya
(%)
(U/mg)
1a (4-Cl-C6H4)
1b (C6H5)
0.21 97(S)
0.46 96(S)
0.21 98(S)
0.01 95(S)
2.30
2.69
1.40
0.18
0.07
3.88
2.61
0.72
1.22
1.15
0.18
0.28
1.35
0.78
99(S)
96(S)
99(S)
98(S)
ND
11.0
5.8
1c (4-CH3-C6H4)
1d (4-iPr-C6H4)
1e (4-tert-Bu-C6H4) 0.03 NDc
6.7
18.0
2.3
1f (4-F-C6H4)
0.44 99(S)
0.14 97(S)
0.14 74(S)
0.40 93(S)
0.09 98(S)
0.01 97(S)
>99(S)
>99(S)
97(S)
98(S)
>99(S)
>99(S)
ND
8.8
1g (4-Br-C6H4)
1h (2-Cl-C6H4)
1i (3-Cl-C6H4)
18.6
5.1
3.1
1j (4-CH3-O-C6H4)
1k (4-CH3S-C6H4)
12.8
18.0
28.0
4.8
Figure 3. Time course of preparative scale biotransformation by
using cell-free extracts of wt SsNIT (A) and its mutant
P194A/I201A/F202V (B).
1l (4-(CH3)2N-C6H4) 0.01
ND
0.28 97(S)
0.01 ND
1m (4-CF3-C6H4)
1n (4-OH-C6H4)
96(S)
ND
78.0
a The activity was determined by the phenol hypochlorite colorimetry
reaction, b ee values were determined by chiral HPLC, c ND means not
detected.
Conclusions
In summary, the activity and enantioselectivity of SsNIT towards
1a was improved by a semi-rational design approach based on
the crystal structures. Several single, double and triple mutants
with superior activity and enantioselectivity have been
identified based on screening assay and combinatorial
mutagenesis. And we successfully identified three key amino
acid residues (194, 201, 202) surrounding the substrate-binding
pocket that affect the steric effects according to docking
experiments. The best mutant P194A/I201A/F202V with a
larger substrate-binding pocket displayed significantly
enhanced catalytic activity and enantioselectivity (S, 99% ee)
toward 1a and other 3-substituted glutaronitriles. In
preparative scale biotransformation of 1a, (S)-2a was obtained
with high time-space productivity, ee value and isolated yield.
Preparative scale biotransformation
(S)-3-(4-chlorophenyl)-4-cyanobutanoic acid ((S)-2a) is
a key
intermediate of (R)-Baclofen and Arbaclofen placarbil, thus the
preparative scale biotransformation of 1a to (S)-2a was performed
by using whole cells of wt SsNIT and its mutant P194A/I201A/F202V
with the substrate concentration of 100 mM at 30ºC. As shown in
Figure 3, 100 mM of 1a was completely hydrolysed by variant
P194A/I201A/F202V within 1h, while wt SsNIT needed 9 h. A little
amide was detected during the reaction and ee value of (S)-2a was
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These results indicate that variant P194A/I201A/F202V is min to measure the activity by phenol hypochlorite colorimetry.
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feasible for application in the production of (S)-2a en route to The by-product and ee values were meDasOuI:r1e0d.10b3y9/cCh8iCraYl02H4P55LCC
(R)-Baclofen.
analysis after the reaction was carried out onvernight.
Preparation of enzymes and activity assay
The wt SsNIT and its mutants were expressed in
Rosetta 2(DE3)pLySs. The recombinant strains were cultured in
the 20 mL of LB medium containing 100 mg/mL ampicillin and
then cultured overnight at 37°C and 200 rpm. The seed broth
was transferred to 800 mL of LB medium containing 100 mg/mL
ampicillin and the culture was grown at 37°C and 200 rpm until
the optical density at 600 nm was between 0.6 to 0.8. 0.1 mM
IPTG was supplemented to the culture to induce protein
expression at 30°C for 10 h. Cells were harvested by
centrifugation (6,000 rpm, 15 min) and washed once with 0.9%
(w/v) NaCl. Proteins were purified through a Ni2+ affinity column
(General Electric Company) and washed using a step-wise
gradient eluent (from 20 to 500 mM imidazole). The purified
enzymes were analyzed by SDS/PAGE and protein
concentration was measured by the BCA Protein Quantitation
Kit.
Experimental section
Materials and methods
3-substituted glutaronitriles were prepared from corresponding
aldehyde and cyanoacetic acid as our previous report.33
DifcoTM LB Broth, Miller (Luria-Bertani) was purchased from
Becton Dickinson and Company. Ampicilin was purchased from
Beijing Probe Bioscience Co., Ltd. Isopropyl β-D-1-
thiogalactopyranoside (IPTG) was purchased from AMRESCO
Inc. Other commercial chemicals were purchased from Aladdin
Industrial Corporation and Sinopharm Chemical Reagent Co.,
Ltd. The restriction enzymes and other reagents for molecular
biology were supplied by Fermentas (Germany) and TaKaRa
(Japan). The conversion, by-product amide and enantiomeric
excess were determined by an Agilent 1200 series high-
performance liquid chromatography (HPLC). H and 13C NMR
1
The standard assay was carried out by mixing 50 µL of 3-(4-
chlorophenyl) glutaronitrile (1a, 200 mM stock in methanol)
with an appropriate amount of enzyme in the sodium
phosphate buffer (100 mM, pH 7.0). The reaction mixture (1 mL,
final volume) was incubated at 30°C for 30 min, and then
stopped by boiling water bath. The precipitated protein was
removed by centrifugation (14000 rpm, 2 min), and supernatant
was subjected to determine the generated ammonia for
assaying the nitrilase activity by the phenol hypochlorite
colorimetry reaction. One unit of the enzyme activity was
defined as the amount of enzyme producing 1 µmol of ammonia
per minute under the standard assay conditions.
spectra were recorded on a Bruker Avance III 600 MHz NMR
spectrometer.
Saturation mutagenesis and site-directed mutagenesis
All mutations of SsNIT were generated in the vector pET-15(b)
with an N-terminal 6×His tag. The site-saturation mutagenesis
(NDT) or site-directed mutagenesis was performed with two-
step PCR strategy.49 A pair of primers for mutagenesis formation
consisted of one non-mutagenic forward primer P1 and one
mutagenic reverse prime (Table S1). The PCR reaction mixture
(50 μL) contained 22 μL water, 1 μL (50~100 ng) template DNA,
1 μL each primers mix (10 μM) and 25 μL of PrimeSTAR Max DNA
Polymeras. The PCR conditions were as follows: 98°C 2 min
(98°C 10 s, 58°C 30 s, 72°C 5 s) × 35 cycles, 72°C 5 min. The
amplified short DNA fragments (ca.300 to 500 bp in length)
were used as megaprimers in the second PCR reaction to
amplify the whole plasmid. The components of PCR reaction
mixture were same as above, except 1 μL amplified short DNA
fragments instead of the primers. The PCR conditions were as
follows: 98°C 2 min (98°C 10 s, 58°C 30 s, 72°C 1 min 10 s) × 30
cycles, 72°C 5 min. The recovered PCR amplicons were purified
and then digested with Dpn I at 37°C for 2 h. Finally, the PCR
products were sequenced and those with mutations were
subjected to transformation for enzyme expression.
Enzymatic Kinetics
The initial-velocity data as a function of substrate concentration
was measured for determination kcat and Km values. Prior to the
reaction, the sodium phosphate buffer (100 mM, pH 7.0) with
different concentration of 1a (0.1–2.0 mM of substrate with
0.15 mL methanol) was incubated at 30°C for 15 min. The
reaction was initiated by addition of purified enzyme (0.05
mg/mL). Enzyme reactions were carried out 3 mL at 30°C, and
the reaction mixtures were stopped with the addition of 0.1 mL
of 6M HCl at 2 to 10 min. HPLC was used for analysis and Origin
8.0 was used for calculation through nonlinear curve fit by
fitting to the equation v=Vmax[S]/([S]+Km). All reactions were
performed in triplicate.
Expression and screening of mutants
The screening of library was performed with 96-well plates
containing wt SsNITas control. Single mutant clones were picked
out from plates and cultivated in 1 mL of LB with ampicillin (100
μg/mL), IPTG (0.1 mmol/L) at 30°C, 800 rpm for overnight. The
cells were washed once with NaCl solution (v/v, 0.9%) and the
reaction was performed with 2 mM 1a at 30°C and 200 rpm for
1 h. The cells were removed by centrifugation (4000 rpm, 10
min), and supernatant was used to screen with the ammonia
HPLC analysis
The reaction was quenched by 6 mol/L HCl. The aqueous
solution was extracted with equal amount of ethyl acetate.
After being dried with anhydrous sodium sulfate, ethyl acetate
was removed and the sample was dissolved in the solvent
mixture of mobile phase. The concentration of product, by-
product and ee value were determined by chiral HPLC analysis
at 30°C on an Agilent 1200 series high-performance liquid
chromatography with CHIRALPAK OD-H column (5 µm, 4.6 mm
× 250 mm) and a UV detector at 210 nm. The flow rate was 0.5
mL/min and the eluent was a mixture of isopropanol, hexane
45
production by phenol hypochlorite colorimetry reaction.44
And then rescreening was performed in a 1 mL reaction with 10
mM 1a, OD600=5 of wet cells and sodium phosphate (100 mM,
pH 7.0) at 30°C and 200 rpm. The reaction was stopped at 30
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and trifluoroacetic acid (30:70:0.1, v/v/v). The product (S)-2a
standard sample was prepared by the hydrolysis of 1a catalyzed
by E. coli whole cells of BjNIT6402 from Bradyrhizobium
japonicum USDA110 according to our previous work.33 The
retention times of (R)-2a, (S)-2a, 1a and rac-amide were 11.3,
12.0, 36.4, 15.9 and 16.5 min, respectively.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The work was financially supported by the National Natural
Science Foundation of China (Grant No. 21572261), Youth
Innovation Promotion Association of the Chinese Academy of
Sciences (Grant No. 2016166) and Tianjin Municipal Science and
Technology
Commission
(15PTGCCX00060
and
15PTCYSY00020). We thank Ms. Jie. Zhang for technical support
with protein purification.
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Catalysis Science & Technology
Page 8 of 8
View Article Online
DOI: 10.1039/C8CY02455C
Improving catalytic efficiency and stereoselectivity of a nitrilase from Syechocystis
sp. PCC6803 by semi-rational engineering en route to chiral -amino acids
Shanshan Yu, Peiyuan Yao, Jinlong Li, Jinhui Feng, Qiaqing Wu, and Dunming Zhu
Simultaneously improving activity and steroselectivity of a nitrilase to catalyze the
desymmetrization of 3-substituted glutaronitriles is presented.