Design of nitrilases with superior activity and enantioselectivity towards sterically hindered nitrile by protein engineering

Article abstract of DOI:10.1002/adsc.201500039

Abstract The enantioselective hydrolysis of ortho-chloromandelonitrile with nitrilase is one of the most attractive approaches to prepare (R)-ortho-chloromandelic acid. To date, efforts to develop this nitrilase-mediated process were plagued by either insufficient ee_p (enantiomeric excess of product) or low activity due to the steric hindrance from the ortho-substituted substrate. To improve the nitrilase potential for producing (R)-ortho-chloromandelic acid, an enhancement of both activity and enantioselectivity towards sterically hindered nitriles would be highly desirable. Molecular docking of the (R)-ortho-chloromandelonitrile into the active site of wild-type 2A6 nitrilase (nitA) allowed the identification of proximal nitA active site residues. Several residues (52, 132, 189 and 190) were selected as targets for single and double point mutation to improve nitA activity and enantioselectivity towards ortho-chloromandelonitrile. Targeted mutagenesis yielded several nitA variants with superior activity and enantioselectivity. The best mutant T132A/F189T exhibited a 4.37-fold higher specific activity (7.39 U/mg) towards ortho-chloromandelonitrile than the wild-type nitA. More importantly, the enantioselectivity (E) was improved from 17.34 to >200, resulting in a highly enantiopure product. Molecular docking experiments further support the enhanced activity and enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of nitA are provided. The amino acids at sites 189 and 132 determine the activity and enantioselectivity towards ortho-chloromandelonitrile. With mutant T132A/F189T as a catalyst, a maximum of 450 mM of (R)-ortho-chloromandelic acid was produced with a 90% conversion and >99% ee_p within 3 h. This is the first time that a high productivity of (R)-ortho-chloromandelic acid of up to 671.76 g L^-1d^-1 using a nitrilase-mediated approach is reported. The engineered T132A/F189T variant represents a promising and competitive biocatalyst for practical application in synthesizing (R)-ortho-chloromandelic acid.

Full text of DOI:10.1002/adsc.201500039

FULL PAPERS
DOI: 10.1002/adsc.201500039
Design of Nitrilases with Superior Activity and Enantioselectivity
towards Sterically Hindered Nitrile by Protein Engineering
a,b
a,b
a,b
a,b
a,b
Ya-Ping Xue, Cheng-Ci Shi, Zhe Xu, Biao Jiao, Zhi-Qiang Liu,
a,b
a,b,
a,b
Jian-Feng Huang, Yu-Guo Zheng, * and Yin-Chu Shen
a
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Peopleꢀs Republic of China
Fax : (+86)-571-8832-0630; phone: (+86)-571-8832-0630; e-mail: zhengyg@zjut.edu.cn
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of
Technology, Hangzhou 310014, Peopleꢀs Republic of China
b
Received: January 25, 2015; Revised: March 16, 2015; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500039.
Abstract: The enantioselective hydrolysis of ortho- importantly, the enantioselectivity (E) was improved
chloromandelonitrile with nitrilase is one of the most from 17.34 to >200, resulting in a highly enantiopure
attractive approaches to prepare (R)-ortho-chloro- product. Molecular docking experiments further sup-
mandelic acid. To date, efforts to develop this nitri- port the enhanced activity and enantioselectivity
lase-mediated process were plagued by either insuffi- shown experimentally and the structural effects of
cient ee (enantiomeric excess of product) or low ac- this amino acid substitution on the active site of nitA
p
tivity due to the steric hindrance from the ortho-sub- are provided. The amino acids at sites 189 and 132
stituted substrate. To improve the nitrilase potential determine the activity and enantioselectivity towards
for producing (R)-ortho-chloromandelic acid, an en- ortho-chloromandelonitrile. With mutant T132A/
hancement of both activity and enantioselectivity to- F189T as a catalyst, a maximum of 450 mM of (R)-
wards sterically hindered nitriles would be highly de- ortho-chloromandelic acid was produced with a 90%
sirable. Molecular docking of the (R)-ortho-chloro- conversion and >99% ee within 3 h. This is the first
p
mandelonitrile into the active site of wild-type 2A6 time that a high productivity of (R)-ortho-chloro-
À1 À1
nitrilase (nitA) allowed the identification of proxi- mandelic acid of up to 671.76 gL d using a nitri-
mal nitA active site residues. Several residues (52, lase-mediated approach is reported. The engineered
132, 189 and 190) were selected as targets for single T132A/F189T variant represents a promising and
and double point mutation to improve nitA activity competitive biocatalyst for practical application in
and enantioselectivity towards ortho-chloromandelo- synthesizing (R)-ortho-chloromandelic acid.
nitrile. Targeted mutagenesis yielded several nitA
variants with superior activity and enantioselectivity.
The best mutant T132A/F189T exhibited a 4.37-fold Keywords: biocatalysis; enantioselectivity; kinetic
higher specific activity (7.39 U/mg) towards ortho- resolution; mutagenesis; nitrilase; protein engineer-
chloromandelonitrile than the wild-type nitA. More ing
Introduction
Resolution procedures include diastereomeric crystal-
[
4]
lization and enantioselective enzymatic hydrolysis of
[
5]
Enantiomerically pure 2-hydroxyarylacetic acids are the O-acetates or esters of hydroxy acids. Neverthe-
valuable intermediates and building blocks for the less, the yield of such approaches is limited to only
synthesis of biologically active compounds and target 50%. The nitrilase-mediated resolution has exciting
[1]
molecules. For instance, (R)-2-chloromandelic acid potential for synthesizing (R)-2-hydroxyarylacetic
(R)-2a] is emerging as the preferred chiral intermedi- acids from nitriles due to the cheap starting material
[
[
6]
ate for the industrial synthesis of the anti-thrombotic and the 100% theoretical yield (Scheme 1). Nitri-
agent (S)-clopidogrel. World-wide sales of (S)-clopi-
[
2]
ACHTUNGTRENNUlGN ases (EC 3.5.5.1) represent a class of hydrolytic en-
dogrel amount to several billion dollars per year. zymes which catalyze the hydrolysis of nitriles to the
Presently, resolution is the most popular way to pre- corresponding carboxylic acids in one single step. The
[
3]
pare chiral 2-hydroxyarylacetic acids in industry.
nitrilase-catalyzed processes to produce carboxylic
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resents a difficult problem for the enzyme design-
[
17]
ers. This is especially the case for the nitrilases to-
wards rac-1a, where it is difficult to implement
a high-throughput method of determining enantiose-
lectivity to allow the rapid screening of a large
number of mutants because of racemization of the
substrate. The task of searching for novel nitrilases
with high activity and excellent enantioselectivity to-
wards sterically hindered nitrile rac-1a still remains
[
18]
a challenge.
Scheme 1. Enantioselective hydrolysis of rac-1 for the pro-
duction of (R)-2 with nitrilase. The residual (S)-1 which is
unreactive to the nitrilase spontaneously decomposed to al-
dehyde plus cyanide, which was then converted to substrate
for the nitrilase.
Here, we have designed several nitrilases with supe-
rior activity and enantioselectivity towards sterically
hindered substrate rac-1a by protein engineering for
the efficient production of (R)-2a with the preselected
2
A6 nitrilase (nitA), which was screened by Robert-
[
19]
son et al.
The molecular docking experiment of
(
R)-1a into the nitA active site was used to identify
acids from nitriles are often preferred to chemical hy- active site residues that are close to the substrate. The
drolysis because the reactions do not require strongly docking experiment identified four positions (52, 132,
acidic or basic reaction conditions and elevated tem- 189 and 190) in the nitA active site that might be the
peratures and produce the desired product with high suitable targets for engineering the acivity and enan-
[
7]
selectivity at high conversion. Over the past de- tioselectivity of nitA towards rac-1a. These positions
cades, an increasing number of studies on nitrilases were randomized by gene site-saturation mutagenesis
has been reported. Nitrilases have been proven to be (GSSM) and four single-site libraries were created.
valuable alternatives to chemical catalysts for the in- Three single mutants at positions 132 and 189 with
dustrial production of fine chemicals such as (R)-man- enhanced nitA activity and enantioselectivity towards
delic acid and pharmaceutical intermediates such as substrate rac-1a were obtained. Then, combinatorial
[
8]
(
R)-ethyl 3-hydroxyglutarate.
Unfortunately, efforts to develop a nitrilase-mediat- were constructed. Several double mutants showed su-
ed hydrolytic process for the economic production of perior activity and excellent enantioselectivity to-
R)-2a from racemic ortho-chloromandelonitrile (rac- wards substrate rac-1a, when compared to the wild-
a) were plagued by either insufficient ee (enantio- type enzyme. A possible mechanism was proposed to
saturation mutagenesis libraries at site 132 and 189
(
1
p
meric excess of product) or low activity. All the exist- explain the experimental observations obtained for
ing nitrilases performed poorly towards the ortho-sub- the wild-type and mutant nitA. This is the first exam-
stituted substrate rac-1a because of the steric hin- ple of enzyme-substrate docking-guided point muta-
drance. Several nitrilases screened from genomic li- tion of the substrate-binding residues to generate
[9]
[10]
braries or engineered from Alcaligenes faecalis
mutant nitrilases with enhanced both activity and
with high enantioselectivity towards rac-1a were often enantioselectivity towards rac-1a. These results pro-
inhibited by the substrate even at a relatively low con- vide some insight into the fundamental question of
centration (10–20 mM). The nitrilases from Alcali- how substrate-binding residues affect substrate bind-
[
11]
genes sp. ECU0401,
1
Labrenzia aggregata DSM ing orientation and thus control the reaction stereose-
[12]
[13]
3394 and Burkholderia cenocepacia J2315 could lectivity.
convert a relative high amount of rac-1a (200–
94 mM) with ee values of 90.4, 96 and 97.6%, re-
4
p
spectively. However, it is still necessary to further im- Results and Discussion
prove their enantioselectivity and activity to meet the
requirement of industrial applications. In our recent Planning of Nitrilase Mutagenesis
work, more than ten nitrilases were discovered by
[
14]
screening different soil samples and several nitrilase The crystal structures of several nitrilase superfamily
genes from phylogenetically distinct organisms were members were well documented and the first crystal
[
15]
cloned and expressed. We also engineered the nitri- structure of Pyrococcus abyssi nitrilase exhibiting true
[
16]
[20]
lase from Alcaligenes faecalis ZJUTB10 to improve nitrilase activity was recently reported. With the in-
its activity towards rac-mandelonitrile (rac-1d). To creased number of the enzyme structures available
our disappointment, none of those nitrilases exhibited from the Protein Data Bank (PDB) and highly devel-
high enantioselectivity and activity towards rac-1a. To oped bioinformatic tools, design of the “hot spots”
date, increasing both the enantioselectivity and activi- which potentially affect the enzymatic activity might
ty of an enzyme reaction for synthetic advantage rep- significantly increase the efficiency of desired mutant
2
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Design of Nitrilases with Superior Activity and Enantioselectivity
[
22]
lase reaction mechanism, Cys162 attacks the cyano
group of nitrile as a nucleophile, Glu46 assumes the
role of a general base, and Lys128 is involved in stabi-
lization of the formed tetrahedral intermediate struc-
ture. The enantioselectivity and activity of nitrilase
should have a close relationship with the spatial con-
formation of catalytic pocket. Several active site resi-
dues (Tyr52, Thr132, Phe189, and Ser190) are in close
proximity to the substrate (R)-1a after a detailed ex-
amination (Figure 1). Both Ser190 and Phe189 were
located at one side of the catalytic tunnel towards the
ortho-chloro substituent and the a-hydroxy group of
the substrate, which might have a considerable effect
on the activity and enantioselectivity towards rac-1a
due to the steric hindrance. The same situation was
observed at Thr132. In addition, the hydroxy in the
Thr132 has a strong hydrogen bond with the a-hy-
droxy in the substrate, which might have an effect on
the enzyme activity. Tyr52 was an important compo-
nent at the bottom of the catalytic tunnel. So four res-
idues (Tyr52, Thr132, Phe189 and Ser190) were select-
Figure 1. Close-up view of the catalytic residues Glu46- ed for mutagenesis.
Lys128-Cys162 (medium grey sticks) and mutated active site
surrounding residues (Tyr52, Thr132, Phe189 and Ser190) of
wild-type (light grey sticks) in complex with the most favor-
Library Construction and Nitrilase Variants Screening
able docked pose for (R)-1a (dark grey sticks). Docking of
substrate was performed using the available tools in Auto-
Dock 4.0.
In order to examine the effect of different amino acid
side chains at the four selected positions (52, 132, 189
and 190) on nitA activity and enantioselectivity, four
single-site libraries were generated by GSSM. The re-
sulting libraries were screened for clones that exhibit-
a semi-rational ed higher activity towards rac-1a. Cells of the mutant
[21]
[11–13,19]
hits. Based on previous work,
design was chosen to engineer nitA to identify hot nitA clones from each library were first grown in 96-
spots for subsequent mutations to increase the activity deep-well plates containing three clones of wild-type
and enantioselectivity. Homology modeling and dock- nitA as control, after which cells were washed, lysed
ing experiments were first performed to gain insights and assayed separately for their ability to hydrolyze
into the binding mode of the (R)-1a with the active rac-1a. The formed products were analyzed by chiral
sites of the reported arylacetonitrilases (Burkholderia HPLC. Overall, 11 out of 744 clones (186 clones for
cenocepacia J2315 nitrilase, Labrenzia aggregate each library) from the four libraries were selected be-
DSMZ 13394 nitrilase, Alcaligenes sp. ECU0401 nitri- cause of their apparent higher activity and enantiose-
lase, A. faecalis ZJUTB10 nitrilase and nitA). lectivity towards rac-1a. The plasmids from the 11 se-
Through a BLAST search against the PDB, the pro- lected clones were isolated, and the gene sequence
tein PH0642 from Pyrococcus horikoshii (PDB acces- analysis showed that the clones contained three muta-
sion code 1J31) showed the highest sequence identity tions (T132R, F189T and F189S). These mutants and
to the reported arylacetonitrilases. The 3D structural wild-type nitA were purified to >90% homogeneity
models of these arylacetonitrilases were constructed using an Ni-based immobilized metal affinity chroma-
based on the crystal structures of protein PH0642. tography procedure. Further evaluation of the activity
The docking experiments showed that nitA was the and enantioselectivity of the purified enzymes was
most suitable candidate for the semi-rational design performed. The results are shown in Table 1. The var-
of nitrilase mutagenesis with higher activity and enan- iants T132R (4.35 U/mg) and F189T (5.87 U/mg) were
tioselectivity towards (R)-2a according to the pro- the best variants with highest activity at site 189 and
[
22]
posed nitrilase reaction mechanism and spatial po- 132. In addition, the enantioselectivities have also
sitions of catalytic pocket and substrate (Supporting been improved greatly. For the second round of
Information, Figure S1).
GSSM, a combinatorial saturation mutagenesis library
The binding mode of the substrate (R)-1a, catalytic at site 132 was constructed by choosing variant F189T
residues and the active site surrounding residues are as a template. Meanwhile, the combinatorial satura-
shown in Figure 1. According to the proposed nitri- tion mutagenesis library at site 189 was constructed
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Table 1. Nitrilase mutants with increased specific activity Kinetic Analysis of Nitrilase Variants
[a]
and enantioselectivity after two rounds of GSSM.
To elucidate the reason for the increase in the activity
from a biochemical perspective, the steady-state ki-
netic parameters of wild-type nitA and its mutants
were measured (Table 2). For the T132R variant,
[
b]
Mutants
Specific activity [U/mg]
eep [%]
None (wild-type)
First round
T132R
F189S
F189T
Second round
T132R/F189C
T132R/F189S
T132R/F189T
T132R/F189H
T132R/F189Y
T132 A/F189T
1.69Æ0.02
86.7
4.35Æ0.09
3.82Æ0.13
5.87Æ0.04
96.0
98.5
>99
a 15% decrease in K combined with a 2.48-fold in-
m
crease in kcat contributed to a total 2.91-fold increase
in k /K . The enantioselectivity (E) for rac-1a was
cat
m
increased from 17.34 to 61.95, when compared to
wild-type nitA. The Km values of F189S and F189T
were close to that of the wild-type nitA, indicating
the similar substrate-binding affinity. The improved
catalytic efficiency (k /K ) is mainly a result of the
5.60Æ0.11
4.63Æ0.01
6.02Æ0.05
3.23Æ0.05
5.87Æ0.12
7.39Æ0.07
>99
>99
>99
>99
>99
>99
cat
m
increase in their turnover frequencies. The mutant
F189T has an excellent enantioselectivity (E>200) to-
wards rac-1a. When the variant site 189 was intro-
duced while T132R as the template, the kcat and cata-
lytic efficiencies of double mutants (T132R/F189C,
T132R/F189S, T132R/F189T, T132R/F189H, and
T132R/F189Y) have not been improved in compari-
son with T132R or F189T. The combination of two
best single mutants T132A and F189T (T132R/F189T)
[
a]
Reaction conditions: 408C, 100 mm Tris-HCl buffer
pH 7.5) containing 5% methanol, 20 mM rac-1a; reac-
(
tion time, 5 min.
Enantiomeric excess of product.
[
b]
by choosing mutant T132R as a template. In total, 28 did not have a synergistic effect on improving enzy-
out of 372 clones (186 clones for each library) from matic activity. However, the enantioselectivities of
the two libraries were selected because of their appar- those variants have been improved greatly when com-
ent higher activity and enantioselectivity towards rac- pared to T132R. When the variant site 132 was intro-
1
a. After gene sequence analysis, six double mutants duced while F189T was the template, the double
(
T132R/F189C, T132R/F189S, T132R/F189T, T132R/ mutant T132A/F189T was found to have the highest
À1
À1
F189H, T132R/F189Y, T132A/F189T) with superior Vmax (8.27 mmolmg min ) and stronger substrate-
activity (>3 U/mg) and excellent enantioselectivity binding affinity. The catalytic efficiency (k /K ) of
cat
m
À1
À1
(
ee >99%) were obtained (Table 1). The double var- mutant T132A/F189T is 150.66 min mM , which is
p
iant T132A/F189T (7.39 U/mg) showed the highest ac- 7.26-fold higher than that of wild-type nitA. The
tivity, which exhibited a 4.37-fold higher specific activ- T132A/F189T variant retained excellent enantioselec-
ity than that of the wild-type nitA (1.69 U/mg).
tivity (E>200).
Table 2. Comparison of the kinetic constants obtained for the wild-type and mutant enzymes.
À1
À1
À1
À1
À1
[a]
Mutant (s)
Substrate
K [mM]
m
Vmax [mmolmg min ]
kcat [min ]
k /K [min mm ]
E
cat
m
None (wild-type)
T132R
F189S
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1b
rac-1c
rac-1d
rac-1b
rac-1c
rac-1d
3.89
3.32
3.79
3.75
3.65
3.60
3.77
3.53
3.41
2.15
5.71
6.23
1.89
2.89
1.76
4.33
2.06
5.12
4.94
7.52
7.08
5.53
7.34
3.15
4.73
8.27
1.18
2.54
8.06
0.29
0.03
2.37
80.68
20.74
60.40
51.05
78.54
75.97
60.16
76.26
34.95
54.33
150.66
8.10
15.97
167.03
3.92
17.34
61.95
162.87
>200
>200
>200
>200
>200
>200
>200
5.70
5.21
150.12
5.62
5.41
134.67
200.53
193.48
294.53
277.30
216.59
287.48
123.38
185.26
323.91
46.22
99.48
315.68
11.36
1.18
92.83
F189T
T132R/F189C
T132R/F189S
T132R/F189T
T132R/F189H
T132R/F189Y
T132A/F189T
None (wild-type)
None (wild-type)
None (wild-type)
T132A/F189T
T132A/F189T
T132A/F189T
0.67
21.44
[
a]
[23]
Enantioselectivity, which was calculated using the following formula
E=ln[1Àc(1+ee )]/ln[1Àc(1Àee )], where c is
p
p
conversion and ee is enantiomeric excess of product.
p
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Design of Nitrilases with Superior Activity and Enantioselectivity
Figure 2. Molecular docking of (R)-1a and (S)-1a (dark grey sticks) into the active site of wild-type (A, B) and mutant
T132A/F189T (C, D). The catalytic residues Glu46-Lys128-Cys162 and mutant sites (132, 189) were shown as medium grey
sticks and light grey sticks, respectively. The dockings of substrates were performed using AutoDock 4.0. The figures were
generated using PyMOL.
We also investigated the effect of the chloro-sub- tioselectivity were observed towards ortho-substituted
stituent position in the substrate on the catalytic per- substrate rac-1a and a general trend can be found
formance and enantioselectivity of wild-type nitA and that the activity and enantioselectivity decrease as the
T132A/F189T variant. For wild-type nitA, the kcat and chloro substituent on the phenyl ring is shifted from
catalytic efficiency towards various substrates with the ortho-position to the para-position.
a chloro substituent in ortho-, meta-, or para-position
in the phenyl ring decrease significantly because of
the steric hindrance. Meanwhile, the enantioselectivi- Characterization of nitA Wild-Type and T132A/
ty also decreased greatly. The substrate without the F189T by Molecular Modeling
substituted group in the phenyl ring (rac-1d) is the
most favorable substrate for the wild-type nitA. For In order to illustrate the differences in activity and
mutant T132A/F189T, the highest activity and enan- enantioselectivity between the wild-type nitA and the
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best mutant T132A/F189T, we modeled the R- and S- mutant, the Cys162 could attack the cyano carbon in
enantiomers of rac-1a into the active site of wild-type both R-isomer and S-isomer as a nucleophile (Sup-
and mutant nitA. According to the proposed nitrilase porting Information, Figure S2), resulting in the
[
22]
reaction mechanism,
the distance between the poorer enantioselectivity. Meanwhile, there are strong
sulfur of the sulfhydryl group on the catalytic Cys162 hydrogen bonds formed between the hydroxy in
and the cyano carbon in the substrate plays a crucial Thr189 and the a-hydroxy in (R)-1b–1d, which could
role in determining the nitrilase catalytic activity to- hinder the release of product and decrease the activi-
wards the substrate. For wild-type nitA, the distances ty.
between the sulfur of sulfhydryl group on the catalytic
Cys162 with the cyano carbon in the substrate (R)-1a
and (S)-1a were 3.79 and 4.09 ꢂ, respectively (Fig- Preparative Application of Mutant Nitrilase for the
ure 2A and B). The Cys162 could attack the cyano Synthesis of (R)-1a
carbon in (R)-1a as well as in (S)-1a as a nucleophile,
resulting in a poor enantioselectivity (E=17.34). In The catalytic property of T132A/F189T was investi-
addition, there are two strong hydrogen bonds be- gated to explore its potential as a candidate biocata-
tween the hydroxy in the substrate (R)-1a and Thr lyst for the resolution of rac-1a. In comparison to iso-
132, which might hinder the release of product, lead- lated nitrilase, whole-cell biotransformation is more
ing to a low activity. For mutant T132A/F189T, the stable due to the presence of their natural environ-
distance between the sulfur of the sulfhydryl group on ment inside the cells. We selected the whole cells of
the catalytic Cys162 and the cyano carbon in (R)-1a is recombinant E. coli BL21(DE3) harboring plasmid
3.37 ꢂ (Figure 2). The nucleophilic attack on this pET-28b (+)-T132A/F189T as biocatalyst. The insolu-
cyano carbon becomes easier by virtue of the proper bility of nitrile substrates in the aqueous reaction mix-
orientation and distance. However, the distance for ture could lead to the mass-transfer problem, thereby
(
S)-1a was 7.25 ꢂ, which is too remote to initiate the decreasing the enzymatic reaction rate.
nucleophilic attack for the sulfhydryl group (Fig- The aqueous-organic biphasic system can enhance
ure 2D). So, the mutant T132A/F189T displayed high the availability of insoluble nitrile substrate to the ni-
activity and enantioselectivity towards rac-1a. The trilase active site, thereby increasing the catalytic effi-
[
12]
result was in agreement with the analysis of kinetics ciency.
The preselected aqueous-organic biphasic
parameters, which implied that the binding affinity of system, toluene-water (2:8, v/v) system, was used in
T132A/F189T to (R)-1a has been significantly im- the enantioselective hydrolysis of rac-1a. The various
proved. The increased activity for T132A/F189T concentrations of substrate from 100 mM to 400 mM
might be caused by the increase in steric bulk intro- were used to evaluate the catalytic activity towards
duced for (R)-1a but not for (S)-1a due to the short rac-1a. The whole cells of recombinant E. coli per-
side chain in Ala132 and Thr189, resulting in a low formed well from 100 mM to 300 mM, giving (R)-2a
steric hindrance towards a chloro substituent at the in >90% conversion and >99% ee . As the rac-1a
p
ortho-position. It benefits the substrate (R)-1a to concentration was increased to 400 mM, little (R)-2a
enter the catalytically active center and change its ori- was produced because the high concentration of ni-
entation to be easily attacked by the sulfur of the sulf- trile was harmful to nitrilase. The enantioselective hy-
hydryl group in Cys162 with a shorter distance. Mean- drolysis with 100 mM of (R)-2a exhibited the highest
while, there is no strong hydrogen bond between the substrate conversion of up to 96.7% (Figure 3). In
a-hydroxy of the substrate with Ala132. The product order to produce (R)-2a at higher substrate conver-
could be released easily, which facilitates the next sion, a fed-batch reaction mode was tried in the pre-
substrate molecule to enter the catalytically active selected toluene-water (2:8, v/v) system. The initial
center. The similar situation was also observed for rac-1a concentration was set at 100 mM. When the
F189T and F189S. For T132R, although Arg132 in this substrate was exhausted in the first batch reaction,
single mutant has a long side chain, its side chain was 100 mM of rac-1a were supplemented per 20 min for
towards the opposite of catalytic pocket and played the fed-batch reaction. Pleasingly, the cumulative (R)-
the same role as Ala132. The mutant T132R enlarged 2a amount reached as high as 450 mM with 90% con-
the pocket and made it possible for Phe189 to be re- version and >99% ee within 3 h (Figure 4). The pro-
p
À1 À1
placed with other residues (Cys, Ser, Thr, His and ductivity was 671.76 gL d without any loss of eep,
Tyr) with higher activity than wild-type nitA. All of which markedly exceeded the average productivity of
À1 À1 [24]
the single mutants and double mutants at 132 and 189 industrial bioprocess (372 gL d ). After recrystal-
obtained in this work showed higher activity and lization in toluene, (R)-2a was obtained as a while
enantioselectivity towards rac-1a than wild-type nitA, solid in 87% isolated yield and >99% ee . Compared
p
implying that the sites 189 and 132 determine the ac- with the existing nitrilase aimed at producing (R)-2a
tivity and enantioselectivity. When substrates (R)-1b– (Table 3), the mutant T132A/F189T-mediated biocata-
1d and (S)-1b–1d were docked into the active site of lytic resolution gave higher substrate loading, enantio-
6
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FULL PAPERS
Design of Nitrilases with Superior Activity and Enantioselectivity
Conclusions
In summary, the activity and enantioselectivity of
nitA towards rac-1a could be improved by a struc-
ture-based rational design approach. Several single
mutants and double mutants with superior nitrilase
activity (>3 U/mg) and enantioselectivity (>99%
ee ) have been identified based on molecular docking
p
experiments, followed by the screening assay based
on the hydrolytic activities towards substrate rac-1a.
The key residues (132, 189) which determine the ac-
tivity and enantioselectivity towards ortho-chloroman-
delonitrile were found. The best mutant T132A/
F189T displays higher activity, productivity and enan-
tioselectivity in the hydrolysis of rac-1a than all the
reported nitrilases, which renders this variant to be
a very suitable candidate for practical application in
the kinetic resolution of rac-1a to (R)-2a, a key inter-
mediate for the synthesis of anti-thrombotic agent
Figure 3. The effect of substrate concentration on conver-
sion and ee . Reaction conditions: 100 mM Tris-HCl buffer
p
(
4
pH 7.5) containing 20% toluene, 0.3 g dry cells, 100–
00 mM (final concentration) rac-1a, 408C.
(
S)-clopidogrel, which is one of the best-selling drugs
for heart attack and stroke treatment. Further studies,
including engineering of nitrilases towards another 2-
hydroxyarylacetonitriles to produce valuable chiral 2-
hydroxyarylacetic acids based on this work, are in
progress.
Experimental Section
Strains, Plasmids and Chemicals
The nitrilase gene (GeneBank: AY487562) with a length of
1
014 bp was synthesized after condons optimization by soft-
ware against E. coli as host according to the method as de-
[15c]
Figure 4. Regioselective hydrolysis of rac-1a for synthesizing
R)-2a with whole cells of recombinant E. coli BL21(DE3)
harboring plasmid pET-28b(+)-T132A/F189T by fed-batch
mode in biphasic system. Reaction conditions: 100 mM Tris-
HCl buffer (pH 7.5) containing 20% toluene, 408C, 0.3 g dry
cells, and 100 mM rac-1a was added per 20 min.
scribed previously.
The restriction sites NcoI and XhoI
(
were introduced at both ends. The ATG in NcoI acted as
the initiation codon with an insertion of Gly at second resi-
dues. E. coli JM109 (Tiangen Biotech. Co. Ltd., Beijing,
China) and plasmid pMD18-T (Takara, Dalian, China) were
used for gene cloning experiments. The BL21 (DE3) (Nova-
gen, USA) and pET-28b(+) (Novagen, USA) were applied
for recombinant protein expression. (R)-2a–2d were pur-
chased from J&K Chemical Co., Ltd. (Shanghai, China).
selectivity, and productivity, making it promising and All the other chemicals used were of analytical grade and
competitive for practical application. commercially available.
Table 3. Comparison on enantioselective hydrolysis of rac-1a with the reported nitrilases.
Entry Biocatalyst Substrate loading [mM] Conversion [%] ee [%] Productivity [gL d ] Ref.
À1 À1
p
[
a]
[9]
1
2
3
4
5
6
Nitrilase 1
pHNIT45
E. coli JM109/pNLE 200
LaN
BCJ2315
T132A/F189T
50
10
92
96
99
90.4
96
97.6
>99
n.a.
[
[
[
[
10]
100
76.5
94.5
84
268.70
171.30
154.4
232.32
671.76
11]
12]
13]
300
494
500
90
this work
[
a]
Not available.
Adv. Synth. Catal. 0000, 000, 0 – 0
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7
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Ya-Ping Xue et al.
FULL PAPERS
Library Construction and Screening
The concentrations of hydrolysis products of rac-1b and
rac-1c were analyzed by HPLC equipped with a Hypersil
ODS (24.6 mmꢃ250 mm, 5.0 mm) column and A215 was mea-
sured. The mobile phase was composed of NH H PO
4
The active site residues at positions 52, 132, 189 and 190
were selected to create saturation mutagenesis libraries. The
corresponding forward and reverse primers (Supporting In-
formation, Table S1) were designed by Primer X. The com-
binatorial saturation mutagenesis library at site 132 was con-
structed using mutant F189T as a template. The combinato-
rial saturation mutagenesis library at site 189 was construct-
ed using mutant T132R as a template. The PCR products
were digested with Dpn I to remove parent plasmid (2 h at
4
2
(
50 mM) and water at a ratio of 35:65 (v/v) with a flow rate
À1
of 1.0 mLmin . The temperature of elution was set at 308C.
For determining the optical purity of the hydrolysis products
of rac-1b and rac-1c, samples were esterified using boron tri-
fluoride in methanol and subjected to chiral HPLC using
a Chiralcel OD column (250 mmꢃ4.60 mm, 5 mm; Daicel
Chemical Industries, Japan) and A228 was measured. The
mobile phase contained n-hexane, isopropyl alcohol and tri-
378C with 10 units of Dpn I), then the reaction mixtures
were transformed into E. coli BL21(DE3) and plated on
fluoroacetic acid (90:10:0.1, v/v) with a flow rate of
À1
Luria-Bertani (LB) medium (Yeast extract, 5 gL ; Peptone,
À1
0
.8 mLmin . The temperature of elution was set at 308C.
À1
À1
1
5
0 gL ; NaCl, 10 gL ; pH 7.0) agar plates containing
For determining the enzyme activity, the reactions were
performed at 408C for 5 min by using a Thermomixer Com-
pact (Eppendorf, Germany). The reaction mixture contained
À1
0 mgmL kanamycin.
All clones containing several parent E. coli were grown in
the respective wells in 96-well plates, in 2.2 mL growblock
(
1.0 mL) Tris-HCl buffer (100 mM, pH 7.5), 20 mM sub-
(
5
Axygen) that contained 1.0 mL LB medium with
0 mgmL kanamycin. The plates were incubated at 378C
strate, 5% methanol and an appropriate amount of pure
enzyme. The reactions were stopped by adding 10 mL HCl
À1
and 200 rpm. When OD600 reached 0.5–0.6, 0.1 mM isopro-
pyl-b-d-1-thiogalactopyranoside (IPTG) was added. Then
the cultivation was continued at 288C for 10 h. Cells were
harvested by centrifugation (48C, 3,000ꢃg, 30 min). The cell
pellets were then resuspended in Bugbuster solution
(
6.0M). The product concentrations were detected by
HPLC. One unit of the enzyme activity (U) was defined as
the amounts of enzyme that produce 1.0 mmol products per
minute under the standard assay conditions.
(
0.5 mL) and the plates were incubated at room temperature
Determinations of Kinetics Parameters
with agitation for 30 min. The hydrolysis reactions (1 mL)
containing Tris-HCl buffer (100 mM, pH 7.5), 20 mM rac-1a,
The kinetic analysis of wild-type and mutant nitA were car-
ried out in Tris-HCl buffer (pH 7.5, 100 mM) at 408C with
purified nitrilases. The final substrate concentration was 0.8–
5% methanol and 100 mL of the cell free extracts were car-
ried out at 408C by using a Thermomixer Compact (Eppen-
dorf, Germany) for 5 min, to which were added 10 mL HCl
3
1
.0 mM and the reactions were stopped by the addition of
0 mL HCl (6.0M). The product concentration of was detect-
(
6.0M). The activity and enantioselectivity of nitrilase were
detected by chiral HPLC as described below.
ed. The values of Km and Vmax were obtained by the Line-
weaver–Burk double-reciprocal method. The value of kcat
was calculated by using the equation kcat =Vmax/[E].
Expression and Purification of Nitrilase Variants
Recombinant E. coli harboring the plasmid pET-28b(+) Molecular Homology Modeling and Docking
containing the wild-type nitA or a mutant nitA gene with
The three-dimensional homology models of nitrilases were
a histidine tag at the carboxyl terminus was grown in LB
À1
generated by the program Modeller 9.12 using crystal struc-
tures of protein PH0642 (PDB accession code 1J31, resolu-
tion 1.6 ꢂ) as template. Docking experiments were per-
formed using the program AutoDock 4.0, and the images
were created using PyMOL. The best quality model generat-
ed by Modeller 9.12 has the highest GA341 score and the
lowest DOPE score. The model was evaluated by Procheck
and Profile-3D (http://www.ebi.ac.uk/thornton-srv/databases/
pdbsum/Generate.html). The nitA showed 25% sequence
identity with the protein from P. horikoshii. The catalytic
triad (Glu46-Cys162-Lys128) of nitA and PH0642 of the
same nitrilase superfamily were completely conserved.
Moreover, the homology model of nitA has a four-layer a-
b-b-a sandwich fold which is similar to other nitrilase super-
family members (Supporting Information, Figure S3). Pro-
check and profile-3D were used to evaluate the modeled
structure. The best quality model of nitA was shown in the
Ramachandran plot, where 82.4% residues were located in
the most favored regions, 13.1% in the additional allowed
regions, and 2.4% in the generously allowed regions. Only
2.1% were located in disallowed regions. Most of the resi-
dues in disallowed regions were located at the end of the
carbon terminal and far away from the catalytic center. The
results from Profile-3D showed that the overall compatibili-
liquid medium containing 50 mgmL kanamycin at 150 rpm
and 378C. Nitrilase expression was induced by adding IPTG
to a final concentration of 0.1 mM when the optical density
reached 0.5–0.6 and then continuously incubated at 288C for
0 h. After centrifugation at 9,000 rpm under 48C for
0 min, the cells were harvested and preserved for further
1
2
experiments.
These mutants and wild-type nitA were produced in the
cytoplasm of E. coli BL21(DE3) and purified to >90% ho-
mogeneity using an Ni-based immobilized metal affinity
The evaluations of activity
and ee were performed by the standard assay with the puri-
[15b]
chromatography procedure.
p
fied nitrilases.
Analytical Methods
The concentration and optical purity of hydrolysis products
of rac-1a and rac-1d were determined by chiral HPLC
equipped with a reverse phase chiral column (Astec Chiro-
biotic R 250ꢃ4.6 mm, Sigma, USA), and A215 was mea-
sured. The mobile phase contained 0.5% AcOH-CH CN
TM
3
À1
(
12:88, v/v) with a flow rate of 1.0 mLmin . The tempera-
ture of elution was 308C.
8
asc.wiley-vch.de
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Design of Nitrilases with Superior Activity and Enantioselectivity
ty score for this model was 107.8 in the scale of the expected
value 69.69–154.87. All these data indicated that the model
of nitA was reliable. Structure-based sequence alignment
was performed using the program Clustal X and the picture
of the sequence alignment was made using the program ES-
PRIPT.
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D
0
yield: 7.3 g (87.0); >99% ee ; [a] : À151.6 (c 0.5, ethanol).
p
[
[
25]
25
D
1
Lit.
[a] : À154.5 (c 0.52, ethanol). H NMR (500 MHz,
D O): d=7.38–7.33 (m, 1H), 7.30 (ddd, J=10.7, 6.0, 3.3 Hz,
2
1
3
1
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9
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1
3
1
84.9996, calcd. for C H ClO [M] : 185; IR (KBr): n=3379,
8
6
3
199, 2927, 1750, 1694, 1477, 1444, 1423, 1264, 1217, 1191,
À1
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 31170761) and the 863 Program
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Design of Nitrilases with Superior Activity and
Enantioselectivity towards Sterically Hindered Nitrile by
Protein Engineering
11
Adv. Synth. Catal. 2015, 357, 1 – 11
Ya-Ping Xue, Cheng-Ci Shi, Zhe Xu, Biao Jiao,
Zhi-Qiang Liu, Jian-Feng Huang, Yu-Guo Zheng,*
Yin-Chu Shen
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
11
ÞÞ
These are not the final page numbers!