Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus tokodaii by site-saturation mutagenesis

Article abstract of DOI:10.1080/10242422.2016.1247823

An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W variant that has excellent selectivity compared with that of wild-type ST0071.

Full text of DOI:10.1080/10242422.2016.1247823

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Improved enantioselectivity of thermostable
esterase ST0071 from archaeon Sulfolobus
tokodaii by site-saturation mutagenesis
Masanaru Ozaki, Norifumi Kawakami, Hiromichi Ohta & Kenji Miyamoto
To cite this article: Masanaru Ozaki, Norifumi Kawakami, Hiromichi Ohta & Kenji Miyamoto
(2016) Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus
tokodaii by site-saturation mutagenesis, Biocatalysis and Biotransformation, 34:5, 249-252,
DOI: 10.1080/10242422.2016.1247823
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Published online: 18 Nov 2016.
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Date: 29 December 2016, At: 10:50

Biocatalysis and Biotransformation, 2016; 34(5):249–252
SHORT COMMUNICATION
Improved enantioselectivity of thermostable esterase ST0071 from
archaeon Sulfolobus tokodaii by site-saturation mutagenesis
MASANARU OZAKI, NORIFUMI KAWAKAMI, HIROMICHI OHTA
& KENJI MIYAMOTO
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama,
Kanagawa, Japan
Abstract
An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but
shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W
variant that has excellent selectivity compared with that of wild-type ST0071.
Key words: Sulfolobus tokodaii; esterase; directed evolution; enantioselectivity
Introduction
resolution of secondary alcohols. Recently,
Angkawidjaja et al. (2012) described the high-reso-
lution crystal structures of ST0071.
Lipases and esterases are important biocatalysts for
industrial applications. They often exhibit high
stereoselectivity and are therefore suitable for the
synthesis of optically active compounds. Many
lipases and esterases have been cloned and char-
acterized; in particular, thermostable hydrolases have
been adopted for industrial use because they exhibit
tolerance to high temperatures and organic solvents.
Previously, we found an esterase, ST0071, from
Sulfolobus tokodaii strain 7 by in silico whole-genome
screening and the enzyme shows high thermostability
(Suzuki et al. 2004). Using ST0071, we successfully
developed a thermally driven domino reaction for the
asymmetric synthesis of (S)-a-arylpropionates from
the corresponding malonic diesters (Wada et al.
2013). We recently reported that ST0071 can
catalyze the asymmetric hydrolysis of bulky malonic
diesters to the corresponding monoesters with excel-
lent enantioselectivity and yield, but it shows very
low enantioselectivity toward various acetates of
secondary alcohols (Wada et al. 2015). Thus, the
active site of ST0071 needs to be redesigned to
improve the enantioselectivity for the kinetic
Directed evolution has emerged as a powerful tool
for improving the catalytic performance of biocata-
lysts and several studies using this method to
improve enzyme enantioselectivity have been
reported (Manco et al. 2002; Park et al. 2005;
Reetz et al. 2007; Kourist and Bornscheuer 2011;
Ema et al. 2012; Tang et al. 2014; Kim et al. 2015).
Protein engineering has contributed to the creation
of enzyme variants with high selectivity for the
preparation of optically active alcohols.
In this study, we first identified amino acid
residues around the substrate-binding site based on
3D structure of ST0071 (Angkawidjaja et al. 2012).
We then carried out site-saturation mutagenesis
based on the homology model and screened a
variant, that showed high enantioselectivity, using
QuickE, which is a spectrophotometric method for
the measurement of selectivity (Janes and Kazlauskas
1997). Finally, we characterized the variant and
compared these results with those of wild-type
ST0071.
Correspondence: Kenji Miyamoto, Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Yokohama, Kanagawa 223-8522, Japan. Tel: +81 455661786. E-mail: kmiyamoto@bio.keio.ac.jp
(Received 14 December 2015; revised 29 June 2016; accepted 4 August 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1247823

250 M. Ozaki et al.
OMe
Materials and methods
AcO
O
O
O
Ph
Chemicals and plasmid
N
O
NO
2
2
1
(R)- and (S)-a-Methoxyphenylacetic acid were
purchased from Tokyo Kasei Kogyo (Tokyo,
Japan). Enantiomerically pure acid and p-nitrophe-
nol were coupled using N,N0-dicyclohexylcarbodii-
mide as a condensation reagent in anhydrous
dichloromethane. Resorufin acetate was purchased
from SIGMA (St. Louis, MO). 1-Phenylethyl alco-
hol, 1-phenyl-1-propanol, 1-phenyl-2-propanol, and
1-phenyl-1-pentanol were purchased from Tokyo
Kasei Kogyo Co. Ltd. (Tokyo, Japan). The alcohol
acetate was synthesized with acetic anhydride and
pyridine. ST0071 mutant genes were inserted into
pET100 vector (Invitrogen, Carlsbad, CA) for pro-
tein expression. B-PER Bacterial Protein Extraction
Reagent was purchased from Thermo Fisher
Scientific Inc. (Yokohama, Japan).
OAc
OAc
Et Ph
OAc
OAc
Me
Ph
n
Ph
Me Ph
Bu
3
4
5
6
Figure 1. Substrates and reference compound.
compound (2) at 572 nm at 25 ꢀC for 1 min
(Figure 1). The QuickE value was calculated using
Equation (1)
vðSÞ=vðrefÞ
vðRÞ=vðrefÞ
Quick E ¼
:
ð1Þ
After isolation of the sequences from selected
positive plasmid clones of the plasmids, the enantios-
electivity of the variant for various compounds was
expressed as the enantiomeric ratio (endpoint E)
(Chen et al. 1982). This value was calculated using
Equation (2).
Library construction and screening for improving
enantioselectivity
Site-saturation mutagenesis with primers bearing an
NNK degenerating codon was used to construct
libraries containing all 20 possible amino acids in
the desired position. A library of 200 clones was
constructed. All clones were expressed in Escherichia
coli BL21 Star (DE3).
eeS
ln½ð1 ꢁ cÞð1 ꢁ eeSÞꢂ
ln½1 ꢁ c1 þ eesꢂ
x ¼
Endpoint E ¼
: ð2Þ
eeS þ eeR
High performance liquid chromatography
(HPLC) was carried out on an LC-2010A HT
system (Shimadzu, Kyoto, Japan) equipped with
chiral column CHIRALCEL OD (4.6 mm
ꢃ250 mm, Daicel Corporation, Tokyo, Japan).
Separation of the enantiomers was achieved using a
flow of 0.5 mL/min at 260 nm, with a mobile phase
composed of hexane–i-PrOH (100:3).
Transformed cells were grown in LB liquid
medium (10 mL) containing 50 mg/mL ampicillin at
37 ꢀC to an OD600 of approximately 0.8. Esterase
expression was induced by the addition of isopropyl-
b-D-thiogalactoside (IPTG; final concentration of
0.1 mM). Cells were incubated for 3 h, and harvested
by centrifugation at 5000g for 10 min, before being
resuspended in B-PER Bacterial Protein Extraction
Reagent (1 mL) and incubated for 15 min at room
temperature. The lysate was centrifuged at 15,000g
for 5 min to remove the insoluble proteins. The
soluble protein solution was incubated at 60 ꢀC for
25 min to denature the proteins other than the
thermostable esterase. The denatured proteins were
removed by centrifugation at 15,000g for 15 min and
the resulting cell-free extract was used to determine
enantioselectivity. QuickE method was used to
determine the enantioselectivity of the variants.
Hydrolyses of the enantiomers of 4-nitrophenyl
2-methoxyphenylacetate (1) liberate the yellow
p-nitrophenol. Resorufin acetate (2) was used as a
reference compound. Reaction conditions were used
as described previously (Janes and Kazlauskas 1997).
We measured the initial rates of hydrolysis of both
enantiomers (1) at 404 nm and the reference
Characterization of enzymes
The endpoint E value was determined at various
temperatures by measuring the enantioselectivity of
the esterase at temperatures ranging from 22 to
80 ꢀC. Thermostability was determined by pre-
incubating purified esterase at 75 ꢀC and analyzing
the residual activity under optimum reaction
conditions.
Enzyme kinetic assay
Enzyme kinetic parameters of ST0071 and variants
were determined from the Lineweaver–Burk plot at
various concentrations of the esters (1) as
substrates.

Improved enantioselectivity of thermostable esterase ST0071
251
Table 1. Kinetic parameters of ST0071 and G274W variant for
Enzyme assay of ST0071 and its variant
ester (1).
The esterase activity against p-nitrophenyl butyrate
(pNP-C4) was determined by measuring the amount
of resulting p-nitrophenol available via enzymatic
hydrolysis. One unit (U) of esterase activity was
defined as the amount of enzyme required to liberate
1 mmol of p-nitrophenol per min. The specific activity
of ST0071 was 165 U/mg proteins at 30 ꢀC.
Configuration
Run Esterase of ester (1) Km (mM) kcat (sꢁ1
Relative
activity (%)
)
1
2
3
4
WT
S
S
R
R
0.54
2.58
5.24
18.1
9.6
125
20.2
100
10
69
3
G274W
WT
G274W
19.5
Table 2. Enantioselectivity of G274W variant at various
temperatures.
Enzyme-catalyzed hydrolysis
The reaction of various esters with ST0071 or the
variant was performed with 10 mM concentration of
the substrate and 450 U of the enzyme in 100 mM
Tris–HCl (pH 8) for acetates of secondary alcohols
in a total volume of 10 mL. After stirring at 30 ꢀC for
1.5–24 h, the reaction mixture was extracted with
diethyl ether. After removal of the solvent in vacuo,
the enantiomeric excess of the substrates and prod-
ucts was then analyzed using HPLC equipped with a
chiral column.
Run Temp. (ꢀC) eeS (%) eeR (%) Conv. (%) Endpoint E
1
2
3
4
5
6
7
22
50
60
65
70
75
80
30.7
98.1
95.0
94.1
87.3
94.8
5.6
99.5
98.3
97.8
96.7
96.3
93.3
59.0
24
50
49
49
48
50
4200
4200
4200
4200
151
106
4.1
8.8
Table 1. The hydrolytic activity (kcat/Km) was
expressed relative to that of (S)-1, taken as 100.
The Km value of the G274W variant for (R)- and (S)-
esters (1) was higher than that of the wild-type
esterase. Similarly, the kcat value of the variant
decreased compared with that of wild-type esterase.
In particular, the kcat value for (R)-1 decreased
compared with the (S)-enantiomer, and S selectivity
might have increased. Gly274 residue is situated
close to His273, a member of the catalytic triad, thus
binding mode by substitution could affect
enantioselectivity.
Results and discussion
Crystal structure of ST0071 (PDB code 3AIK)
provided the basis for the identification of six
amino acid residues around the substrate binding
site. The catalytic triad components (Ser150,
Asp243, and His273) were well conserved.
Residues Tyr89, Phe197, Leu198, His202, Asp204,
and Gly274 are situated within 3 around the
substrate. These positions were independently
altered by mutagenic polymerase chain reaction
with primers bearing the NNK codon. A library of
200 clones (single and double mutants) was con-
structed and all clones were expressed in E. coli.
After partial purification using B-PER, the enantios-
electivity of all variant esterases was confirmed by the
QuickE method. The enantiomers of 4-nitrophenyl
2-methoxyphenylacetate (1) were used to determine
the QuickE value. ST0071 could not selectively
hydrolyze the ester (1); therefore, we expected that it
would be easy to detect any improved enantioselec-
tivity of the variant. From the results of screening,
the selectivity of most variants appeared equal to or
lower than that of the wild-type enzyme (data not
shown). However, the G274W variant had a higher
QuickE value (3.38 ꢄ 0.34, S selectivity) compared
with wild-type ST0071 (1.56 ꢄ 0.14, S selectivity).
To clarify the enantioselectivity of wild-type esterase
and the G274W variant, the Michaelis constant (Km)
and catalytic center activity (kcat) values were
determined by double-reciprocal plots. The kinetic
constants for the (R)- and (S)-esters (1) are shown in
Next, we conducted G274W variant-catalyzed
kinetic resolutions of 1-phenylethyl acetate (3) at
various temperatures and evaluated the enantios-
electivity based on the endpoint E values (Table 2).
G274W showed an excellent endpoint E value
(4200) at 22 ꢀC (Run 1) in contrast to wild-type
ST0071 (E ¼ 11). Below 75 ꢀC, the variant showed
excellent selectivity (Runs 2–6). At 80 ꢀC, the E
value dramatically decreased (Run 7, E ¼ 4), because
of thermal denaturation of the variant.
The endpoint E values at 50 ꢀC of various
secondary alcohol acetates with the G274W variant
are summarized in Table 3. Wild-type ST0071 did
not show enantioselectivity at 50 ꢀC (data not
shown), although the variant showed excellent
enantioselectivity toward 1-phenylethyl acetate
(Run 1, E ꢅ 200) and 1-phenyl-1-propanol acetate
(Run 2, E ꢅ 200). When the alkyl group was
changed to a propyl (Run 3), the enantioselectivity
greatly decreased (E ¼ 4.5). The variant can catalyze
the hydrolysis of 1-phenyl-2-propanol acetate (6) in
moderate selectivity (Run 4, E ¼ 36). In all cases, the
endpoint E values at 50 ꢀC of G274W were higher
than that of wild-type esterase at 20 ꢀC.

252 M. Ozaki et al.
Table 3. Substrate specificity of G274W variant.
G274Wa
cc (%)
Wild-type ST0071b
Run
Substrate
Time (h)
Endpoint E
Time (h)
cc (%)
Endpoint E
1
2
3
4
3
4
5
6
1.5
1.5
24
50
17
3.6
9.1
4200
4200
4.5
2
2
2
2
40
32
8.6
12
11
16
3.6
3.2
1.5
36
aReaction temperature, 50 ꢀC.
bReaction temperature, 20 ꢀC.
cConversion calculated from c ¼ eeS/(eeS +eeR).
Ema T, Nakano Y, Yoshida D, Kamata S, Sakai T. 2012.
Redesign of enzyme for improving catalytic activity and
enantioselectivity toward poor substrates: manipulation of the
transition state. Org Biomol Chem 10:6299–6308.
Janes LE, Kazlauskas RJ. 1997. Quick E. A fast spectrophotomet-
ric method to measure the enantioselectivity of hydrolases.
J Org Chem 62:4560–4561.
Kim J, Kim S, Yoon S, Hong E, Ryu Y. 2015. Improved
enantioselectivity of thermostable esterase from Archaeoglobus
fulgidus toward (S)-ketoprofen ethyl ester by directed evolution
and characterization of mutant esterases. Appl Microbiol
Biotechnol 99:6293–6301.
The thermostability of the esterases was studied at
75 ꢀC by measuring residual activity with pNP-C4 as
the substrate. The activity of G274W decreased to
20% after 60 min incubation. The G274W variant
retained 85% activity after incubation at 70 ꢀC for
60 min. From these results, we were able to confirm
that the G274W variant exhibited sufficient thermo-
stability for industrial use.
Kourist R, Bornscheuer UT. 2011. Biocatalytic synthesis of
optically active tertiary alcohols. Appl Microbiol Biotechnol
91:505–517.
Conclusions
We successfully isolated variant G274W, which
acted on various acetates of secondary alcohols.
Additionally, this variant showed excellent enantios-
electivity compared with the wild-type ST0071.
Thus, G274W variant is a useful tool for the
synthesis of pharmaceuticals and naturally occurring
compounds with a chiral center.
Manco G, Carrea G, Giosue E, Ottolina G, Adamo G, Rossi M.
2002. Modification of the enantioselectivity of two homologous
thermophilic carboxylesterases from Alicyclobacillus acidocaldar-
ius and Archaeoglobus fulgidus by random mutagenesis and
screening. Extremophiles 6:325–331.
Park S, Morley KL, Horsman GP, Holmquist M, Hult K,
Kazlauskas RJ. 2005. Focusing mutations into the P. fluorescens
esterase binding site increases enantioselectivity more effectively
than distant mutations. Chem Biol 12:45–54.
Reetz MT, Puls M, Carballeira JD, Vogel A, Jaeger K-E, Eggert T,
Thiel W, Bocola M, Otte N. 2007. Learning from directed
evolution: further lessons from theoretical investigations into
Declaration of interest
cooperative mutations
ChemBioChem 8:106–112.
in
lipase
enantioselectivity.
The authors report no conflicts of interest. The
authors alone are responsible for the content and
writing of this article.
Suzuki Y, Miyamoto K, Ohta H. 2004. A novel thermostable
esterase from the thermoacidophilic archaeon Sulfolobus tokodaii
strain 7. FEMS Microbiol Lett 236:97–102.
Tang L, Su M, Chi L, Zhang J, Zhang H, Zhu L. 2014. Residue
Val237 is critical for the enantioselectivity of Penicillium
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