Directed evolution of nitrilase PpL19 from Pseudomonas psychrotolerans L19 and identification of enantiocomplementary mutants toward mandelonitrile

Article abstract of DOI:10.1016/j.bbrc.2015.11.038

Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis combined with site-directed mutagenesis was performed to iden

Full text of DOI:10.1016/j.bbrc.2015.11.038

Biochemical and Biophysical Research Communications 468 (2015) 820e825
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Directed evolution of nitrilase PpL19 from Pseudomonas
psychrotolerans L19 and identification of enantiocomplementary
mutants toward mandelonitrile
*
**
Huihui Sun, Hualei Wang , Wenyuan Gao, Lifeng Chen, Kai Wu, Dongzhi Wei
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-
mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis
combined with site-directed mutagenesis was performed to identify the key residues responsible for
nitrilase enantioselectivity. Five enzyme mutants exhibiting distinct selectivity were generated and four
Received 23 October 2015
Accepted 7 November 2015
Available online 11 November 2015
“hot spots” (M113, R128, A136, and I168) responsible for enantioselectivity toward mandelonitrile were
Keywords:
identified and characterized. Furthermore, through saturation mutagenesis, positions 113 and 128 were
confirmed to substantially influence the enantioselectivity of PpL19, and certain replacements of the
methionine at position 113, in particular, were found to reverse the enantioselectivity of PpL19 from S- to
R-selectivity. Two other single mutants of the enzyme, PpL19-A136Y and -I168Y, also showed reversed
selectivity and preferentially produced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively). By
combining the beneficial mutations, two enantiocomplementary nitrilase mutants, PpL19-LH and PpL19-
GYY, were created, which exhibited high S- and R-selectivity toward mandelonitrile, respectively: PpL19-
LH showed the highest S-selectivity toward mandelonitrile ever reported (91.1% ee), and, notably, the
PpL19-GYY mutant was identified to be highly R-selective (90.1% ee) and thus an unexpected enantio-
complementary mutant for mandelonitrile.
Directed evolution
Enantiocomplementary
Nitrilase
Pseudomonas psychrotolerans L19
©
2015 Elsevier Inc. All rights reserved.
1. Introduction
inflammatory drugs such as celecoxib and deracoxib [5].
Although several enantioselective nitrilases have been reported
to date, most of these enzymes show high selectivity toward (R)-
mandelonitrile [6]. The nitrilase-mediated pathway has been suc-
cessfully applied in industry by Lonza, BASF, and Mitsubishi Rayon
to produce (R)-(-)-mandelic acid from (R,S)-mandelonitrile in
amounts reaching at least several tons annually. However, no
analogous system for the synthesis of the (S)-enantiomer has been
reported. To the best of our knowledge, only one published study
has described the existence of S-selective nitrilases that exhibit low
preference (<30% enantiomeric excess (ee)) for the formation of (S)-
mandelic acid from racemic mandelonitrile [7]. Moreover, in
another recent study, mutants of nitrilase from Pseudomonas fluo-
rescens EBC191 exhibiting reversed enantioselectivity were ob-
tained that generated (S)-mandelic acid from mandelonitrile;
however, the ee values of none of the mutants were >37%, and all
mutants generated the byproduct mandelamine in different pro-
portions [8].
In recent years, nitrilase-mediated biocatalysis has attracted
substantial attention from scholars and entrepreneurs. Nitrilase-
mediated highly enantioselective synthesis of (R)-mandelic acid
and its derivatives has been extensively studied, and increasing
interest has been devoted to this bioprocess because it can poten-
tially provide 100% theoretical yield in a dynamic kinetic resolution
manner (Fig. 1) [1,2]. As key intermediates, (R)-(-)-mandelic acid is
widely used in the preparation of semi-synthetic penicillin and
cephalosporin and also used as a universal chiral resolving agent to
resolve racemic alcohols and amines in the industry [3,4]; (S)-
(þ)-mandelic acid can be used for synthesizing nonsteroidal anti-
*
Corresponding author.
** Corresponding author.
E-mail addresses: hlwang@mail.ecust.edu.cn (H. Wang), dzhwei@ecust.edu.cn
Previously, we identified a novel S-selective nitrilase, PpL19,
(
D. Wei).
http://dx.doi.org/10.1016/j.bbrc.2015.11.038
006-291X/© 2015 Elsevier Inc. All rights reserved.
0

H. Sun et al. / Biochemical and Biophysical Research Communications 468 (2015) 820e825
821
Fig. 1. Enantioselective hydrolysis of mandelonitrile to (R)-(ꢂ)-mandelic acid mediated by a nitrilase.
from Pseudomonas psychrotolerans L19: It could enantioselectively
hydrolyze racemic mandelonitrile into (S)-mandelic acid with an ee
value of 52.7% and no byproduct was observed [9]. In this study, we
aspired to identify the key residues responsible for the enantiose-
lectivity of PpL19; identification of these residues can help guide
the engineering of nitrilase enantioselectivity. Moreover, it is
currently of considerable interest to generate nitrilase mutants that
can convert mandelonitrile to (S)-mandelic acid exhibiting high
optical purity.
pellets were resuspended in phosphate buffer (100 mM, pH 7.0)
and supplied with 10 mM mandelonitrile to initiate the hydrolysis
reaction. The reaction mixtures were incubated at 30 C on a
ꢀ
rotary shaker (200 rpm) for 12 h, quenched by adding 1/10 (v/v)
2 M HCl, and centrifuged at 10,000 ꢁ g for 20 min. The obtained
supernatants of the reaction mixture were screened using the
Berthelot assay [11]; the mutants that had lost their activity to-
ward mandelonitrile were efficiently eliminated at this step. The
reaction mixtures containing the positive mutants were sequen-
tially analyzed using chiral HPLC in order to search for mutants
displaying enhanced enantioselectivity toward mandelonitrile.
2
. Materials and methods
2.1. Materials
2.4. Construction of nitrilase mutants through site-directed
mutagenesis
The nitrilase gene (PpL19) from P. psychrotolerans L19 was syn-
thesized by Shanghai Generay Biotech Co., Ltd. [9], and it was
ligated to the expression plasmid pET28a(þ) to generate the re-
combinant plasmid pET28a/PpL19. Escherichia coli BL21 (DE3) was
used as the host for nitrilase protein expression. Mandelonitrile and
mandelic acid were purchased from Trademax Pharmaceuticals &
Chemicals Co., Ltd. (Shanghai, China).
Amino acid substitutions were introduced by using the Quick-
Change™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)
according to supplier instructions. The recombinant plasmid
pET28a/PpL19 was used as the template for the mutagenesis re-
action. Mutagenic primers were designed using Stratagene's web-
based QuickChange® Primer Design Program (available online at
http://www.stratagene.com/qcprimerdesign). The primers con-
taining the appropriate base changes are listed in Supplementary
Table 1.
2.2. Random mutagenesis and library construction
The expression plasmid pET28a/PpL19 carrying the nitrilase
gene from P. psychrotolerans L19 was used as the template for the
mutagenesis reactions. Error-prone PCR was performed by using an
Instant Error-Prone PCR Kit according to the manufacturer's in-
2
.5. Construction of nitrilase mutants through site-saturation
mutagenesis
0
structions (TIANDZ, Beijing, China). The primers used were 5 -
0
0
Site-saturation mutagenesis at the selected positions was
CGCGGATCCATGAAAGCCCTTCACAG-3 and 5 -CCCAAGCTTAGTCC-
GAAAAGTCGTCG-3'. The amplified DNA fragments were digested
with BamHI and HindIII, whose target sites were incorporated into
the primers (underlined), and the resulting fragments were ligated
into pET28a(þ) digested with the same restriction enzymes. Sub-
sequently, E. coli BL21 (DE3) was transformed with the ligation
mixture.
performed using the QuickChange™ Site-Directed Mutagenesis
Kit and degenerate oligonucleotides. The oligonucleotide se-
quences are listed in Supplementary Table 1. The target amino
acid positions were coded by NNK, where N ¼ A, G, T, or C and
K ¼ G or T.
2.6. Enzyme assay
2.3. High-throughput screening
The activities of PpL19 and its mutants were measured (at
ꢀ
High-throughput screening was performed in 96-deep-well
30 C) in 0.5-ml reaction mixtures that contained sodium phos-
phate butter (pH 7.0, 100 mM), mandelonitrile (10 mM), and
20 mg of wet cells. The reactions were quenched by adding 0.1 ml
of 2 M HCl when the mandelonitrile was exhausted. Lastly, the
mixtures were centrifuged and the supernatants were subjected
to HPLC analysis.
plates. The individual clones were cultured in ZYM-5052 auto-
inducing medium supplemented with the appropriate concen-
ꢀ
tration of kanamycin for 36 h at 20 C (with shaking at 150 rpm)
ꢀ
[
10]. The cells were harvested through centrifugation at 4 C for
10 min and then washed with physiological saline. The obtained

822
H. Sun et al. / Biochemical and Biophysical Research Communications 468 (2015) 820e825
2.7. Kinetic resolution of racemic mandelonitrile
two mutantsdB29 and D78dthat demonstrated complementary
R-selectivity (ee values: 66.7% and 74.3%, respectively). To identify
the mutation sites, DNA sequencing was performed, and six mu-
tation positions were identified in the five mutants. The mutants A5
and D96 shared the same mutation site, M113, but featured distinct
amino acid substitutions: the Met at this position was replaced by
Gln in A5 and Cys in D96. Besides, there was one other mutation
P199Q in the D96 mutant. Sequence analysis also identified two
mutations (R128H and G114L) in E68 mutant, and further revealed
that B29 and D78 contained the mutations A136Y and I168Y,
respectively, which reversed the enantioselectivity in favor of R
products at high ee values (>66%).
Racemic mandelonitrile was hydrolyzed using wild-type PpL19
and the mutants as follows: appropriate amounts of wet cells
exhibiting equal enzyme activity and 10 mM racemic mandeloni-
trile (diluted from a 200 mM stock solution in methanol) were
ꢀ
mixed and incubated at 30 C on a rotary shaker (200 rpm). The
reactions were terminated by adding 2 M HCl (50 ml) when the
substrate was exhausted, and the cells were removed by means of
centrifugation (10,000 ꢁ g, 20 min); the ee values were then
measured through HPLC analysis.
2.8. Analytical methods
3
.2. Identification of amino acid substitutions that caused a change
The turnover of nitriles was routinely quantified through HPLC
in enantioselectivity
analysis. The amounts of mandelonitrile and its product were
determined by using a Zorbax SB-Aq column (4.6 ꢁ 250 mm, 5 m)
Agilent Technologies) at a flow rate of 1 ml/min with an eluting
m
To identify the specific mutation positions responsible for the
enantioselectivity of PpL19, site-directed mutagenesis was per-
formed to generate single mutations. Seven mutations were
generated individually and the mutants' enantioselectivity toward
mandelonitrile was determined. As shown in Table 1, the mutations
M113Q, M113C, and R128H contributed to the enhancement of the S
enantioselectivity of PpL19: the ee values of the product were >60%.
Moreover, we identified two mutations, A136Y and I168Y, respon-
sible for the R-selectivity, and these mutants preferentially pro-
duced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively).
However, the mutations G114L and P199Q in clones E68 and D96,
respectively, did not affect the enzyme's enantioselectivity and
were therefore considered to be neutral.
(
solvent system of phosphoric acid (0.1%, v/v) and methanol (40:60,
v/v) and measuring the absorbance at 210 nm (A210). The enan-
tiomers of mandelic acid were separated by using a reverse chiral
phase column (CHIRALCEL OZ-3R, 4.6 mm ꢁ 250 mm, 5
mm) (Daicel
Chemical Industries, Ltd.) at a flow rate of 0.4 ml/min with an
eluting solvent system of acetic acid (0.1%, v/v) and isopropanol
30:70, v/v); once again, the A210 values were measured.
(
3
. Results
3
.1. Screening and characterization of positive clones from a
random-mutation library
3
.3. Saturation mutagenesis
The gene encoding nitrilase PpL19 was mutated randomly
through error-prone PCR performed using an Instant Error-Prone
PCR Kit. The digested PCR fragment and a large fragment of
pET28a(þ) were ligated and transformed into E. coli BL21(DE3) to
generate a library of randomly mutated nitrilases. To express the
nitrilase, we successfully used an auto-induction strategy, which
can substantially reduce the workload and the chance of
contamination by elimination the process of IPTG addition. The
Berthelot assay was first used to determine the activity of the
mutants by measuring the release of ammonium during the re-
action; at this step, the mutants that had lost activity toward
mandelonitrile were eliminated. The reaction mixtures in which
nitrilase activity was detected were analyzed using chiral HPLC in
order to search for mutants displaying enhanced enantiose-
lectivity toward mandelonitrile.
Because the three independent mutations (M113Q, M113C,
R128H) at positions M113 and R128 enhanced the S enantiose-
lectivity of PpL19 toward mandelonitrile, we performed saturation
mutagenesis at these two positions in order to identify the effect of
distinct amino acid residues on the enzyme's enantioselectivity and
further improve the enzyme's S-selectivity toward mandelonitrile.
The results showed that the enantioselectivity of the mutants
harboring various substitutions at site 113 differed widely (Table 2).
Table 2
The enantioselectivity of the mutants based on site-maturation mutagenesis at
selected sites.
Mutations
M113
R128
After screening approximately 8000 clones, we identified three
mutants (A5, D96, E68) that showed higher S enantioselectivity
toward mandelonitrile (ee values: 60.5%e62.8%) than did the wild-
type enzyme (ee value: 52.7%) (Table 1). Notably, we also identified
ee (%)
Enantio-preference
ee (%)
Enantio-preference
A
C
D
E
40.3
60.6
72.1
62.1
ND
58.5
ND
47.9
ND
75.3
52.7
0
ND
60.5
ND
S
S
S
S
ND
e
e
ND
e
F
Table 1
G
H
I
K
L
M
N
P
Q
R
S
R
S
e
The enantioselectivity of the nitrilase PpL19 mutants based on random mutagenesis
and site-directed mutagenesis.
63.9
e
S
S
44.9
e
Mutant
Mutation
ee(%)
Mutation)
ee (%)
(Mutant)
Enantio-preference
S
S
(
e
WTa
A5
D96
e
52.7
60.5
60.6
52.3
63.9
51.7
66.7
74.3
52.7
60.5
60.9
S
S
S
S
S
S
R
R
e
M113Q
M113C
P199Q
R128H
G114L
A136Y
I168Y
e
S
e
52.7
e
S
E68
62.8
28.9
17.9
29.8
ND
R
S
S
T
e
B29
D78
66.7
74.3
V
W
Y
e
e
66.9
S
e
a
The nitrilase PpL19 was designated as the wild type WT.

H. Sun et al. / Biochemical and Biophysical Research Communications 468 (2015) 820e825
823
Besides the initially identified mutants M113Q and M113C, four
other M113 mutants (M113D, M113E, M113L, M113Y) showed
higher enantioselectivity than did wild-type PpL19. Among these,
the M113L mutant exhibited the highest S-selectivity (75.3%).
Furthermore, some of the mutations decreased the enantiose-
lectivity considerably, and in the case of two mutations (M113G,
M113S), it was even reversed to R-selectivity, with ee values of
location of the four “hot spots,” multiple-sequence alignment was
conducted on nitrilase PpL19 with the seven microbial nitrilases
and eleven members of the nitrilase superfamily for which struc-
tural information was available [15]. The results showed that the
positions at which the mutations occurred, M113, R128, A136, and
I168, did not participate in the formation of the PpL19 spiral
structure, because they were not present within the interaction
areas such as the “A,” “C,” “D,” and “E” surfaces (Supplementary
Fig. 1). To further analyze these positions, a structural model of
PpL19 was also constructed as described in our previous study [9].
The first two “hot spots,” M113 and R128, were located near the
active site of the enzyme (<5 Å) (Fig. 2). Recent biochemical and
modeling studies on the nitrilase from Rhodococcus rhodochrous
ATCC 33278 showed that the residues at positions 114 and 129 in
this enzyme, which correspond to the positions 113 and 128 in
PpL19, were located within the substrate-binding pocket [13]. And
rational design of enzymes typically focused on introducing mu-
tations within the substrate-binding sites in order to improve
enzyme properties such as enantioselectivity [16e18]. Conversely,
the residues A136 and I168dwhose mutation to Tyr inverted
selectivity toward the R-enantiomerdwere located far from the
active site (>10 Å) (Fig. 2). Thus, the mutations of the two positions
were supposed not to inteact directly with the substrate, but may
influence the structure of binding pocket through synergistic effect
that caused a different binding mode compared with the WT. Most
of the results obtained in the directed evolution experiments also
agree with this finding [19e21]. In directed evolution, the target is
to mutate all the amino acids equally, and this can create a large
number of distant mutations that are also critical for controlling the
enzyme's enantioselectivity; therefore, directed evolution can
generate increased numbers of beneficial mutants.
2
8.9% and 58.5%. These results demonstrated that the amino acid at
position 113 is an essential residue responsible for the enantiose-
lectivity of nitrilase PpL19.
With regard to site 128, previous studies had demonstrated that
the enzyme was active only when positively charged amino acids
(
Arg, Lys, or His) were present at this position [12,13]. Thus, in this
study, only a few mutations featuring distinct characteristics were
generated at this site, including R128A (neutral amino acid), R128E
(
negatively charged amino acid), and R128K (positively charged
amino acid). The mutants R128A and R128E displayed no activity
toward mandelonitrile (Table 2), which agreed with previous
findings [12,13]. However, the mutant R128K demonstrated low
enantioselectivity (44.9% ee) as compared with the wild-type
enzyme (52.7% ee) and the R128H mutant (63.9% ee).
3.4. Combination of mutations
The effects of mutations are often additive, and combinations of
mutations are therefore frequently used for enhancing the perfor-
mance of enzymes [14]. The aforementioned results revealed that
the mutations M113L and R128H greatly increased the S-enantio-
selectivity of PpL19 toward mandelonitrile. Thus, the double-
mutant PpL19-LH containing both of these mutations was gener-
ated through site-directed mutagenesis. The enantioselectivity of
the mutant was enhanced, with the product being generated at
Molecular docking experiments were performed to gain insights
into the enantioselectivity of nitrilase PpL19 from structural aspects
presented in our previous study [9]. The hydrolysis of nitrile sub-
strates is initiated through nucleophilic attack on the cyano carbon
atom by the S atom of C164 and subsequently form a covalent
thioimidate intermediate. Thus the distance between the sulfur of
Cys and the cyano group of mandelonitrile was crucial for the ef-
ficiency of the hydrolysis. The distinct hydrolysis rates exhibited
91.1% ee. Furthermore, we created another double-mutant, PpL19-
YY, by combining the A136Y and I168Y mutations, and this
mutant produced (R)-mandelic acid with an ee value of 89.7%, an R-
enantioselectivity higher than that obtained with the single mu-
tations A136Y and I168Y. Given that the mutant M113G generated
through site-saturation mutagenesis at position 113 also displayed
reversed enantioselectivity and favored the R product (58.5% ee)
(Table 2), we generated the triple-mutant A136Y/I168Y/M113G in
order to further improve the R-selectivity of PpL19 toward man-
delonitrile. The result showed, as expected, that the R-selectivity of
this mutant was markedly enhanced (to 90.9% ee). Collectively, our
results demonstrated that the double-mutant PpL19-LH (M113L/
R128H) and the triple-mutant PpL19-GYY (M113G/A136Y/I168Y)
generated here displayed the highest enantioselectivity among our
tested mutants and favored of the S and R products, respectively.
4
. Discussion
In this study, we performed directed evolution on the S-selective
nitrilase PpL19 from P. psychrotolerans L19 that can hydrolyze
racemic mandelonitrile to (S)-mandelic acid with an ee value of
5
2.7%. We have identified four crucial positions (“hot spots”) in the
sequence space of nitrilase PpL19 that strongly influence the
enantioselectivity toward mandelonitrile. Among the mutations of
the four “hot spots,” the mutation I168Yda single amino acid
exchangeddecreased and even reversed the selectivity from S to R
(
with an ee value of 74.3%). Moreover, the replacement of the
methionine at position 113 with a leucine or aspartate residue and
the substitution mutation R128H resulted in a substantial increase
in enantioselectivity.
Most active nitrilases are widely recognized to form spiral
structures that consist of 4e22 subunits, and this process could
influence the activity and stability of the nitrilases. To identify the
Fig. 2. Localization of the identified “hot spots” in the structural model of nitrilase
PpL19. The amino acid residues M113, R128, A136 and I168 are shown with carbon
atom in cyan. The conserved catalytic triad (E48, K130, C164) are shown with carbon
atom in magenta. The sulphur, oxygen and nitrogen atoms are shown in yellow, red,
and blue, respectively. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

824
H. Sun et al. / Biochemical and Biophysical Research Communications 468 (2015) 820e825
Fig. 3. Docking poses of (S)-mandelonitrile in the binding pockets of the mutant PpL19-LH (A) and mutant PpL19-GYY (B) and of (R)-mandelonitrile in the binding pockets of the
mutant PpL19-LH (C) and mutant PpL19-GYY (D). Hydrogen bonds were indicated with blue dash lines. Distances between (S)-mandelonitrile or (R)-mandelonitrile and Cys-164
were marked with black plain lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
toward R- and S-mandelonitrile result in the enantio-preference of
the nitrilase. We docked both enantiomers of mandelonitrile into
the active site of the nitrilase in an effort to understand the drastic
change of the enantioselectivity between the two enantiocomple-
mentary mutants obtained (Fig. 3).
mandelonitrile (3.661 Å) was less than that in the wild-type
enzyme (3.833 Å), and the distance in the case of (S)-mandeloni-
trile was beyond the suitable range (4.358 Å) (Fig. 3C and D). These
findings fully agree with the results of the enantioselectivity
analysis.
When the two enantiomers of mandelonitrile were docked in
the active site of the S-selective nitrilase mutant PpL19-LH, large
differences were observed (Fig. 3A and B). In the PpL19-LH mutant,
the distance between the cyano group of (S)-mandelonitrile and
the sulfur of Cys (3.450 Å) was shorter than that in the wild-type
enzyme (3.751 Å). By contrast, in the PpL19-LH mutant docked
with (R)-mandelonitrile, the distance between the catalytically
active Cys164 and the cyano group was considerably longer
This study has laid the foundation for further improving the
mandelic acid enantioselectivity of nitrilases, and this can poten-
tially lead to the development of novel biocatalysts for the pro-
duction of (S)- and (R)-mandelic acid. Future analyses of mutant
nitrilases that exhibit substantially enhanced enantioselectivity
should broaden our understanding of the principles underlying
enzymatic hydrolysis of racemic mandelonitrile.
(
4.083 Å) than that in the wild-type enzyme (3.833 Å). The shorter
Acknowledgments
distance toward (S)-mandelonitrile and longer distance toward (R)-
mandelonitrile compared with those of the WT contributed to the
enhancement of PpL19-LH mutant S-selectivity. Furthermore, a
hydrogen bond was observed between (S)-mandelonitrile and
Trp165 in this mutant, which could help the substrate in a proper
orientation and stabilize the tetrahedral intermediate. However, no
hydrogen bond with any amino acid was detected when (R)-man-
delonitrile was docked, which can account for the enzyme's ten-
dency to bind to the S-enantiomer of the substrate. This finding also
contributed to the enhanced S-selectivity of the PpL19-LH mutant
for mandelonitrile.
This work was supported by the National Natural Science
Foundation of China (No. 21406068/B060804), a China Postdoctoral
Science Foundation funded project (No. 2014M560308), and Na-
tional Major Science and Technology Projects of China
(2012ZX09304009).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2015.11.038.
In the R-selective PpL19-GYY mutant, we found that (R)-man-
delonitrile was engaged in the hydrogen bonding required for
effective catalysis, but no hydrogen bond was formed in this mutant
with the disfavored substrate (S)-mandelonitrile. Moreover, the
distance for the nucleophilic attack of the catalytic triad toward (R)-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bbrc.2015.11.038.

H. Sun et al. / Biochemical and Biophysical Research Communications 468 (2015) 820e825
cultures, Protein. Expr. Purif. 41 (2005) 207e234.
825
References
[11] M.W. Weatherburn, Phenol-hypochlorite reaction for determination of
ammonia, Anal. Chem. 39 (1967) 971e974.
[
[
[
1] A. Banerjee, P. Kaul, U.C. Banerjee, Purification and characterization of an
[
12] S.J. Yeom, H.J. Kim, J.K. Lee, D.E. Kim, D.K. Oh, An amino acid at position 142
in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the
substrate specificity for aliphatic and aromatic nitriles, Biochem. J. 415
enantioselective arylacetonitrilase from Pseudomonas putida, Arch. Microbiol.
1
84 (2006) 407e418.
2] Zhang ZhijuCn, Yu Huilei, T. Imanaka, Xu Jianhe, Efficient production of (R)-
-)-mandelic acid by isopropanol-permeabilized recombinant E. coli cells
expressing Alcaligenes sp. Nitrilase, Biochem. Eng. J. 95 (2015) 71e77.
3] Liu Zhiqiang, Dong Lizhu, Cheng Feng, Wang Yuanshan, Ding Jienv,
Zheng Yuguo, Shen Yinchu, Gene cloning, expression, and characterization of
a nitrilase from Alcaligenes faecalis ZJUTB1, J. Agric. Food Chem. 59 (2011)
(
2008) 401e407.
(
[
[
13] S.J. Yeom, J.K. Lee, D.K. Oh, A positively charged amino acid at position 129 in
nitrilase from Rhodococcus rhodochrous ATCC 33278 is an essential residue for
the activity with meta-substituted benzonitriles, FEBS, Lett. 584 (2010)
106e110.
14] R. Fujii, Y. Nakagawa, J. Hiratake, A. Sogabe, K. Sakata, Directed evolution of
Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity,
Protein. Eng. Des. Sel. 18 (2005) 93e101.
11560e11570.
[
4] Zhu Dunming, C. Mukherjee, E.R. Biehl, Hua Ling, Discovery of a mandeloni-
trile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome
mining, J. Biotechnol. 129 (2007) 645e650.
5] C. Mateo, A. Chmura, S. Rustler, F.V. Rantwijk, A. Stolz, R.A. Sheldon, Synthesis
of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase
bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activ-
ity, Tetrahedron Asymmetry 17 (2006) 320e323.
6] Wang Huilei, Sun Huihui, Wei Dongzhi, Discovery and characterization of a
highly efficient enantioselective mandelonitrile hydrolase from Burkholderia
cenocepacia J2315 by phylogeny-based enzymatic substrate specificity pre-
diction, BMC, Biotechnol 13 (2013) 14e24.
7] D.E. Robertson, J.A. Chaplin, G. DeSantis, M. Podar, M. Madden, E. Chi,
T. Richardson, A. Milan, M. Miller, D.P. Weiner, K. Wong, J. McQuaid, B. Farwell,
L.A. Preston, Tan Xuqing, M.A. Snead, M. Keller, E. Mathur, P.L. Kretz, M.J. Burk,
J.M. Short, Exploring nitrilase sequence space for enantioselective catalysis,
Appl. Environ. Microbiol. 70 (2004) 2429e2436.
8] O. Sosedov, A. Stolz, Improvement of the amides forming capacity of the
arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed
mutagenesis, Appl. Microbiol. Biotechnol. 99 (2015) 2623e2635.
9] Sun Huihui, Gao Wenyuan, Fan Haiyang, Wang Hualei, Wei Dongzhi,
Expression, characterization of a novel nitrilase PpL19 from Pseudomonas
psychrotolerans L19 with S-selectivity towards mandelonitrile present in
active inclusion bodies, Biotechnol. Lett. (2015), http://dx.doi.org/10.1007/
s10529-015-1992-0.
[
15] R.N. Thuku, D. Brady, M.J. Benedik, B.T. Sewell, Microbial nitrilases: versatile,
spiral forming, industrial enzymes, J. Appl. Microbiol. 106 (2009) 703e727.
16] M. Ivancic, G. Valinger, K. Gruber, H. Schwab, Inverting enantioselectivity of
Burkholderia gladioli esterase EstB by directed and designed evolution,
J. Biotechnol. 129 (2007) 109e122.
[
[
[
[
[
17] A. Magnusson, K. Hult, M. Holmquist, Creation of an enantioselective hydro-
lase by engineered substrate-assisted catalysis, J. Am. Chem. Soc. 123 (2001)
[
[
4354e4355.
18] D. Rotticci, J.C. Rotticci-Mulder, S. Denman, T. Norin, K. Hult, Improved
enantioselectivity of a lipase by rational protein engineering, ChemBioChem.
2
(2001) 766e770.
19] G.P. Horsman, A.M.F. Liu, E. Henke, U.T. Bornscheuer, R.J. Kazlauskas, Muta-
tions in distant residues moderately increase the enantioselectivity of Pseu-
domonas fluorescens esterase toward methyl 3-bromo-2-methylpropanoate
and ethyl 3-phenylbutyrate, Chem. Eur. J. 9 (2003) 1933e1939.
[
[
[
20] K. Liebeton, A. Zonta, K. Schimossek, M. Nardini, D. Lang, B.W. Dijkstra,
M.T. Reetz, K.E. Jaeger, Directed evolution of an enantioselective lipase, Chem.
Biol. 7 (2000) 709e718.
21] S. Park, K.L. Morley, G.P. Horsman, M. Holmquist, K. Hult, R.J. Kazlauskas,
Focusing mutations into the P. fluorescens esterase binding site increases
enantioselectivity more effectively than distant mutations, Chem. Biol. 12
[
(2005) 45e54.
[10] F.W. Studier, Protein production by auto-induction in high-density shaking