Characterization of a novel esterase isolated from intertidal flat metagenome and its tertiary alcohols synthesis

Article abstract of DOI:10.1016/j.molcatb.2012.04.015

A gene coding for an esterase (EstEH112) was isolated from metagenome originated from Korean intertidal flat sediment. The putative esterase gene encoded a 340 amino acids protein with characteristic residues of lipolytic enzymes such as a conserved pentapeptide (GXSXG), the typical catalytic S-D-H triad, and a GGG(A)X-motif in the oxyanion hole near the active site similar to the hormone sensitive lipase (HSL) family. p-Nitrophenyl butyrate was the best substrate for the enzyme among the other p-nitrophenyl esters investigated. The apparent optimal temperature and pH for EstEH112 was 35°C and at pH 8.0, respectively. EstEH112 efficiently catalyzed the hydrolysis of various large tertiary alcohol esters. These characteristics of EstEH112 make it a potential candidate for application in biocatalysis.

Full text of DOI:10.1016/j.molcatb.2012.04.015

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of a novel esterase isolated from intertidal flat metagenome
and its tertiary alcohols synthesis
Ki-Hoon Oha, Giang-Son Nguyenb, Eun-Young Kima, Robert Kouristc, Uwe Bornscheuerb,
Tae-Kwang Oha, Jung-Hoon Yoond,∗
a Industrial Biotechnology & Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yuseong, Daejon, Republic of Korea
b Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff Str. 4, 17487 Greifswald, Germany
c Institute of Chemistry of Biogenic Resources, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany
d Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A gene coding for an esterase (EstEH112) was isolated from metagenome originated from Korean inter-
tidal flat sediment. The putative esterase gene encoded a 340 amino acids protein with characteristic
residues of lipolytic enzymes such as a conserved pentapeptide (GXSXG), the typical catalytic S-D-H
triad, and a GGG(A)X-motif in the oxyanion hole near the active site similar to the hormone sensitive
lipase (HSL) family. p-Nitrophenyl butyrate was the best substrate for the enzyme among the other p-
nitrophenyl esters investigated. The apparent optimal temperature and pH for EstEH112 was 35 ◦C and at
pH 8.0, respectively. EstEH112 efficiently catalyzed the hydrolysis of various large tertiary alcohol esters.
These characteristics of EstEH112 make it a potential candidate for application in biocatalysis.
© 2012 Elsevier B.V. All rights reserved.
Received 20 December 2011
Received in revised form 17 April 2012
Accepted 17 April 2012
Available online 24 April 2012
Keywords:
Metagenome
Esterase
HSL family
Tertiary alcohol synthesis
1. Introduction
ester bonds. Esterases prefer to hydrolyze the ester bond of short
chain triglyceride or ester whereas lipases catalyze the hydrolysis
The metagenomic approach is based on the construction of
a genetic material library directly obtained from environmental
sources and is one of the simplest ways of exploring the enor-
mous natural genetic space for a better understanding of the
microbial community in various environments and for finding new
biocatalysts or compounds useful for the biochemical and phar-
maceutical industries. Through screening based on functions or
sequence homologies, many unique enzymes have been isolated
from metagenome originated from diverse environments such
as soil, water, sediment, and extreme habitats [1–3]. Intertidal
flat sediments are affected by dynamic physicochemical condi-
tions such as periodic floodtides, a higher degree of change in
the salinity, and water temperature. These additional variables in
the environment cause a remarkable and unique bacterial diver-
sity in these types of sediments compared to other marine sources
[4]. Therefore, metagenome libraries from intertidal flat sediments
include diverse and valuable genetic materials encoding biocata-
lysts including lipases and esterases [5,6].
of triglyceride bearing long-chain fatty acids. These enzymes are
␣/␤-hydrolase fold enzymes and contain the catalytic triad (Ser-
Asp(Glu)-His) and an oxyanion hole. The serine residue is generally
located in a conserved Gly-X-Ser-X-Gly pentapeptide. Microbial
lipolytic enzymes have been divided into eight families (Family
I–VIII) [7]. Recently further subgroups were suggested based on the
lipolytic enzymes isolated from metagenomic libraries from marine
environment including intertidal flat sediment [5,6], surface sea
water [8], and deep sea sediment [9].
Since many of lipases and esterases exhibit broad substrate
specificity, position selectivity, and stereoselectivity, they serve as
useful biocatalysts in order to obtain especially optically pure sec-
ondary alcohol synthesis and primary alcohol synthesis [10,11].
Similarly, optically pure tertiary alcohols (TAs) are also widely
used as building blocks for synthesis of various organic chemicals
and valuable pharmaceuticals. For instance, linalyl alcohol, which
is found in many flowers and spices, is one of the most impor-
tant terpene alcohols in fragrance and flavor industry. The tertiary
␣-acetylenic alcohols 2-phenylbut-2-yn-2-ol and 1,1,1-trifluoro-2-
phenylbut-3-yn-2-ol were intermediates for the synthesis of A2A
receptor antagonists that were orally active in a mouse catalepsy
model [12] or the acetylene moiety of 2-pyridylbut-3-yn-2-ol can
be used to synthesize useful heterocyclic structures [13]. Chemical
synthesis of optically pure TAs generally requires harsh conditions
Esterases (EC 3.1.1.1), as well as lipases (EC 3.1.1.3), are a
group of carboxyesterases catalyzing the cleavage and formation of
∗
Corresponding author. Tel.: +82 31 290 7800; fax: +82 31 290 7816.
E-mail address: jhyoon69@skku.edu (J.-H. Yoon).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.015

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K.-H. Oh et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
[14]. On the other hands, the synthesis using enzymatic biotrans-
formation gives an opportunity to make more environmentally
friendly synthetic routes. Esterases [15] as well as lipases [16], pro-
teases [17], halohydrin dehalogenases [18], or epoxide hydrolases
[19] were reported for the synthesis of TAs through kinetic resolu-
tion of racemates. However, tertiary alcohol esters are not suitable
substrates for most of esterases and lipases. Recently, the GGG(A)X-
motif in oxyanion hole was found important for the activity toward
tertiary alcohols, though a GX-type lipase from Burkholderia cepa-
cia was able to catalyze the enantioselective hydrolysis of tertiary
cyanohydrins [17,20]. Lipase from Candida rugosa, pig liver esterase,
acetylcholine esterases, recombinant esterase from Bacillus subtilis,
and some esterases from metagenome possessing the GGG(A)X-
motif catalyzed the reaction toward tertiary alcohol with various
enantioselectivity [21–23].
In this study, we explored a metagenomic library constructed
from intertidal flat sediment at Saemankum in the west coastal
region of Korea to isolate a new hormone sensitive lipase like
esterase from the family IV using a catalytic activity based screen-
ing. The cloning, expression, and determination of biochemical
properties of this recombinant esterase and its activity toward ter-
tiary alcohols are reported in this paper.
unless stated otherwise. The synthesis of tertiary alcohol esters
(TAEs) 1a–1h was performed as described [22,24,25] (Fig. 1).
2.2. Methods
2.2.1. DNA extraction from environmental samples and
construction of the metagenomic library
A metagenome sample from the intertidal flat sediments of the
Korean western coastal region in Saemankum was extracted as
previously described [26]. The environmental genomic DNAs were
resolved in 1% (w/v) agarose by pulsed-field gel electrophoresis
with a CHEF-DRIII system (Bio-Rad, USA) in order to fraction-
ate the approximately 35-kb DNA. A metagenomic library was
constructed using a CopyControlTM fosmid library production
kit (Epicentre, USA) according to the manufacturer’s instruc-
tions.
2.2.2. Screening of a lipase/esterase gene and subcloning of
positive clone
Activity-based esterase/lipase screening was performed
by plating the transformants onto Luria-Bertani (LB) agar
plates containing chloramphenicol (12.5 ␮g/mL) and 1%
(v/v) emulsified tributyrin. The plasmid designated pFos-
Lip was isolated from the Escherichia coli transformant
that showed the largest halo. The pFosLip was partially
2. Materials and methods
digested with Sau3AI, ligated with
a pUC19 vector, and
transformed into E. coli DH5␣. The colony with the halo
was selected and sequenced. The open reading frame of
lipase/esterase was defined using the Lasergene 6.0 (DNASTAR
Inc., USA).
2.1. Chemicals
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany),
Fig. 1. Kinetic resolution of tertiary alcohol acetates with EstEH112.

K.-H. Oh et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
69
2.2.3. Expression and purification of the esterase
[ꢀ] = [ꢀ]obs × mrw/10lC, where [ꢀ]obs is the ellipticity measured in
degrees, mrw is the mean residue molecular mass, C is the pro-
tein concentration in g/L, and l is the optical pathlength of the cell
(in cm). The thermal denaturation curve was fitted as two-state
transition model [27].
The full length of the putative esterase gene was
amplified by PCR with two primers (5ꢀ-GGAATTCCATATG-
AGGTGAATCTATGACAAGC-3ꢀ and 5ꢀ-CCGCTCGAGAATTTATTG-
TAGGTGCTGCA-3ꢀ; underlined letters indicate the NdeI and XhoI
recognition sites, respectively). The amplified DNA fragment was
digested with NdeI and XhoI, and was then ligated with plasmid
pET-22b(+) (Novagen, Germany). The resulting recombinant plas-
mid, designated as pET-estEH112, was used to transform E. coli
DH5␣. Once the sequence had been confirmed, the expression
was done by introducing the recombinant plasmid into E. coli
C43(DE3). The recombinant, E. coli EstEH112, was cultured in
an LB medium containing ampicillin (50 ␮g/mL) at 37 ◦C. The
EstEH112 expression was induced by adding 0.5 mM isopropyl-
␤-d-thiogalactopyranoside (IPTG) at an absorbance of ∼0.6 at
600 nm before cultivation for 12 h at 21 ◦C. Cells were harvested
by centrifugation and resuspended in 50 mM Tris–HCl buffer (pH
8.0) containing 300 mM NaCl. Cells were then disrupted by soni-
cation and were centrifuged in 28,000 × g at 4 ◦C for 30 min. The
supernatant was applied to a nickel–nitrilotriacetic acid (Ni–NTA)
column (QIAGEN GmbH, Germany). After washing with a buffer
(5 mM imidazole; 300 mM NaCl; 50 mM Tris–HCl buffer, pH 8.0)
the bound esterase was eluted using 250 mM imidazole in the same
buffer. The collected proteins were loaded onto a Superdex200 Gel
Filtration Column (GE Healthcare, USA) equilibrated with 20 mM
Tris–HCl buffer (pH 8.0) containing 150 mM NaCl. The column
was eluted with 0.5 mL/min of an equilibrium buffer using a
BioLogic DuoFlow Chromatography System (Bio-Rad Laboratories,
USA). Fractions exhibiting esterase activity were analyzed using
SDS-PAGE.
2.2.5. Effect of detergents, metal ions, and organic solvents on the
activity of EstEH112
The effects of detergents on the esterase activity were analyzed
by measuring the remaining activities through additions of 0.1%
and 1% (v/v) of various detergents in 100 mM Tris–HCl buffer (pH
8.0) into the enzyme solution. The effect of divalent metal ions on
the activity of EstEH112 was compared by adding 1, 5, and 10 mM
of CaCl2, CuSO4, MgSO4, FeSO4, ZnSO4, NiSO4, MnSO4, or CoCl2.
In order to estimate the organic solvent tolerance of EstEH112,
1 mg/mL of enzyme solutions were incubated in 10, 30, and 50%
(v/v) of water-miscible organic solvents such as dimethylsulfoxide
(DMSO), dimethylformamid (DMF), acetonitrile, ethanol, acetone,
and isopropanol, and water-immiscible organic solvents such as
ethyl acetate, n-butanol, chloroform, toluene, xylene, and n-hexane
for 1 h at 35 ◦C with shaking at 1400 rpm using a Thermoshaker
(Eppendorf, Germany). The treated enzyme solutions were 50-
fold diluted in 100 mM Tris–HCl, pH 8.0. The residual activity of
EstEH112 relative to non-solvent containing enzyme solution was
measured. All assays were performed in three times independently
under standard assay condition.
2.2.6. General procedure for esterase-catalyzed kinetic resolution
100 ␮g of the enzyme with 13.5 mg of racemic linalyl acetate
in 1 mL of 100 mM Tris–HCl buffer, pH 8.0 was mixed on a shak-
ing incubator (rpm 200) at 35 ◦C for 12 h. The reaction mixture was
diluted by four times volume of ethanol and analyzed by thin layer
chromatography using Merck silica gel 60 F254 (Merck, Germany)
plates and petroleum ether/ethyl acetate (5:1) as eluent. Com-
pounds were visualized by spraying with vanillin/concentrated
sulfuric acid (5 g/L) followed by heat treatment [28].
In order to determine the catalytic activity of EstEH112 on the
synthetic tertiary alcohol esters by 1a–i, 12 ␮mol of each ester was
dissolved in 100 ␮L co-solvent and 900 ␮L esterase solution [with a
crude esterase concentration of 1–3 mg/mL dissolved in phosphate
buffer (100 mM, pH 7.5)] was added to form a total volume of 1 mL.
The reaction mixtures were shaken in a Thermoshaker (Eppendorf,
Germany) at 37 ◦C for a certain time (0.5, 1, 4 and 24 h), after which
a 300 ␮L sample was taken. The sample was extracted twice with
400 ␮L dichloromethane. The combined organic layers were dried
over anhydrous Na2SO4, filtered and transferred to a GC-vial. Enan-
tioselectivity and conversion were calculated according to Chen
et al. [29].
2.2.4. Biochemical characterization of the EstEH112
The enzyme concentration was determined by the Bradford
assay (BioRad, USA) using bovine serum albumin as standard. The
standard assay was carried out for 4 min at 35 ◦C using reaction mix-
ture consisted of 20 ␮L of 100 mM p-nitrophenyl butyrate (pNPB)
in ethanol, 40 ␮L of ethanol, and 0.94 mL of 100 mM Tris–HCl
buffer (pH 8.0) containing an appropriate amount of the enzyme,
unless otherwise specified. The amount of p-nitrophenol liberated
during the reaction was kinetically measured by its absorbance
at 405 nm using a DU800 spectrophotometer (Beckman, USA)
and using a molar extinction coefficient of 17,000 M−1 cm−1. One
unit of enzyme activity was defined as the amount of enzyme
needed to release 1 ␮mol of p-nitrophenol per min at 35 ◦C. Sev-
eral p-nitrophenyl esters (acetate, propionate, butyrate, caproate,
caprylate, caprate, and laurate) were used for the determination
of substrate specificity. Kinetic constant of EstEH112 was deter-
mined using non-linear regression analysis in Origin 8.5 (Microcal,
USA). The optimum pH for enzyme activity was determined at 35 ◦C
from pH 4.0 to 11.0 in 100 mM GTA buffer by measuring liber-
ated p-nitrophenol at 348 nm (isosbestic point of p-nitrophenol).
pH stability of esterase was determined by incubating the enzyme
in 100 mM GTA buffer (pH 4–11) for 1 h at 25 ◦C, and relative activ-
ity was estimated at 35 ◦C. To determine optimum temperature, the
reaction mixture was incubated for 10 min at various temperatures
(10–55 ◦C). Thermostability of esterase was determined by prein-
cubating for 1 h at 10–60 ◦C in 20 mM Tris–HCl buffer, pH 8.0 with
150 mM NaCl. Subsequently, the residual activity was measured at
35 ◦C.
2.2.7. Chiral gas chromatography analysis
Chiral GC-analyses were performed by using a Hydrodex-␤-3B
(Column A), Hydrodex-␤-TBDAc (Column B) and Hydrodex-␥-
TBDAc (Column C) from Machrey-Nagel (Düren, Germany) on a
GC-2010 gas chromatograph (Shimadzu, Tokyo, Japan). Chiral anal-
ysis of 1a–h was performed as described [22,24,25].
2.2.8. Nucleotide sequence accession number
The nucleotide sequences obtained were deposited in the Gen-
Bank database under accession number EU515238.
Thermal denaturation curve of EstEH112 were obtained from
molar ellipticity change of 0.16 mg/mL of EstEH112 in 20 mM
Tris–HCl buffer, pH 8.0 at 222 nm using a JASCO J-815 (Jasco,
Japan) with 1 cm of pathlength cuvette. The temperature was grad-
ually increased by 1 ◦C/min and signal changes were measured
and averaged from three independent experiments. Molar ellip-
ticity per mean residue, [ꢀ], was calculated from the equation:
3. Results
3.1. Screening and analysis of putative esterase sequence
A DNA library from a sediment sample collected from an
intertidal flat sediment was constructed using a fosmid vector,

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K.-H. Oh et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
Fig. 2. Conserved amino acids sequence and phylogenetic analysis of EstEH112. (A) Multiple alignment of the partial amino acid sequences containing the conserved motifs
of the pentapeptide GXSXG and the putative catalytic triad residues of the esterases from the metagenome (EU515238), fls1 gene from South China Sea Marine Sediment
belonging to Hormone Sensitive Lipase (HSL) family (ACL67387) [9], esterase from marine sediment (ADH59414), and esterase from Marine gamma proteobacterium HTCC2143
(EAW33037). Symbols: (᭹) GGG(A)X-motif in oxyanion hole; (*) amino acid residues belonging to the catalytic triad. (B) Phylogenetic tree of EstEH112 and closely related
proteins. Phylogenetic analysis was performed using the Megalign in Lasergene 6.0 (DNASTAR Inc., USA). The alignment of amino acids sequence was carried out by Clustal
V. Except for EstEH112 and related proteins, the protein sequences for families of bacterial lipolytic enzymes previously classified by Arpigny et al. [7] and novel lipases from
marine environmental metagenomes (Lee et al. [5], Kim et al. [6], Jeon et al. [31]) were retrieved from GenBank (http://www.ncbi.nlm.nih.gov).
pCC1FOS. This was followed by screening for lipolytic activ-
ity on a tributyrin agar plate. As a result, a positive clone
showing the highest lipolytic activity from approximately 6000
colonies was selected and sub-cloned. The sequence analysis of
the insert DNA of the selected sub-clone, which gave halo for-
mation showed the presence of one open reading frame (ORF)
consisting of 1020 nucleotides that encode for a protein (EstEH112)
family IV through phylogenetic analysis (Fig. 2B). These results
show that EstEH112 belongs to the HSL family.
3.2. Biochemical characteristics of the recombinant EstEH112
In order to investigate the biochemical properties, EstEH112
was over-expressed with a six-histidine tag fused at its C terminus
in E. coli and purified to homogeneity. Specificity of the enzyme
was investigated using p-nitrophenyl esters (pNPEs) of varying
chain-length as substrates. The esterase showed hydrolyzing activ-
ity toward a wide range of pNPEs (C2–C12) and the highest activity
was found for p-nitrophenyl butyrate (pNPB) (Fig. 3). These results
indicated that EstEH112 is an esterase not a lipase. The kinetic
parameters, Km and kcat, for EstEH112 were determined using
pNPB as a substrate over a concentration range of 0.05–3 mM by
non-linear regression analysis using the Michaelis–Menten equa-
tion (Fig. S1). The Km, kcat, and kcat/Km values of EstEH112 for pNPB
were 0.92 0.07 mM, 23.0 1.0 s−1, and 25.1 s−1 mM−1, respec-
tively.
of 340 amino acids with
a molecular mass of 37 kDa. The
BLAST search against the NCBI non-redundant protein database
(http://www.ncbi.nlm.nih.gov/BLAST/) revealed that the deduced
amino acid sequence of the ORF was similar to several predicted
lipolytic enzymes annotated as esterase from various cultured or
uncultured bacteria such as the fls1 gene from South China Sea
Marine Sediment belonging to the hormone sensitive lipase (HSL)
family (GenBank accession no. ACL67387, identity 62%) [9], an
esterase from marine sediment (GenBank accession no. ADH59414,
identity 50%), and an esterase from Marine gamma proteobacterium
HTCC2143 (GenBank accession no. EAW33037, identity 48%). In
the amino acid sequence of EstEH112, the catalytic triad, Asp281,
His311, and catalytic nucleophile serine at position 185 in the con-
sensus GXSXG pentapeptide, and GGG(A)X motif at the positions
117–120 in the oxyanion hole which are characteristic for the HSL
family could be found (Fig. 2A). EstEH112 was also clustered in
The optimal conditions for the activity of the enzyme were mea-
sured at a temperature range of 10–60 ◦C and at a pH range of
6–11 using p-nitrobutyrate as substrate. EstEH112 showed max-
imal activity at 35 ◦C and pH 8.0, respectively (Fig. 4A and B).

K.-H. Oh et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
71
Fig. 3. Substrate specificity and specific activity toward pNPEs and natural sub-
strate of the EstEH112. The esterase activity of the purified EstEH112 enzyme
was assayed toward various pNPEs at 25 ◦C and pH 8.0. Substrates used were p-
nitrophenyl acetate (C2), p-nitrophenyl propionate (C3), p-nitrophenyl butyrate (C4),
p-nitrophenyl caproate (C6), p-nitrophenyl caprylate (C8), p-nitrophenyl caprate
(C10), and p-nitrophenyl laurate (C12). The error bars represents means SD (n = 3).
EstEH112 retained the 30% of its maximum activity at 10 ◦C. The
enzyme was stable up to 40 ◦C after incubation at each tempera-
ture for 1 h (data not shown). Also, the thermal denaturation curve
showed that EstEH112 started to unfold around 40 ◦C and was fully
denatured above 60 ◦C (Fig. 4C). The melting temperature of this
enzyme was determined to 54.8 0.2 ◦C.
3.3. Effects of various detergents, metal ions, and organic solvents
on the activity of EstEH112
The effects of various detergents and divalent metal ions on the
EstEH112 were also examined. 1% detergent already severely inhib-
ited the hydrolysis of pNPB by the enzyme (Table S1). Most divalent
metal ions had little effect on the enzyme activity except ferric ions
which inhibited the activity to about 40% in 5 mM FeSO4 (Table S2).
These results indicate that divalent metal ions are unnecessary for
the catalytic activity of EstEH112.
In order to test the organic solvent tolerance of EstEH112, vari-
ous organic solvents added in enzyme solution at concentration of
10, 30, and 50% (v/v). In 10% water-miscible organic solvents, the
enzyme activity retained or was only slightly decreased. However,
Table 1
Effects of various organic solvent on EstEH112 activity.
Organic solvents
log Pa
Residualactivity(%)b atconcentration(%, v/v)of
Fig. 4. Properties of EstEH112. (A) Effect of temperature on enzyme activity in pH
8.0. (B) pH effect on enzyme activity (ꢀ) and stability (᭹). (C) Thermal denaturation
curve of EstEH112. The error bars represents means SD (n = 3).
10%
Water-miscible organic solvents
30%
50%
DMSO
DMF
Acetonitrile
Ethanol
Acetone
Isopropanol
−1.35
−1.01
−0.34
−0.31
−0.24
0.05
95.8 5.0
88.6 4.4
71.0 4.3
87.8 6.3
99.0 2.7
90.4 3.5
105.8 2.0
30.3 2.6
5.9 0.9
58.5 4.0
2.4 0.9
5.9 0.7
22.7 0.5
1.6 0.7
5.7 2.3
2.5 1.1
0.5 0.8
4.8 1.4
higher concentration of organic solvents caused dramatic deacti-
vation of the enzyme with the exception using DMSO. The addition
of 10% n-butanol, toluene, and xylene caused a rapid decrease of
the enzyme activity (Table 1).
Water-immiscible organic solvents
Ethyl acetate
n-Butanol
Chloroform
Toluene
0.73
0.88
1.97
2.73
3.12
64.5 5.9
3.7 2.4
76.2 4.4
16.0 0.3
5.9 1.7
38.7 2.2
5.0 2.1
6.3 2.2
15.9 1.4
6.3 3.2
3.4 2.0
3.4 2.0
5.2 1.6
3.0 0.1
5.0 0.1
3.4. The activity of EstEH112 toward tertiary alcohols
For the activity toward tertiary alcohols, linalyl acetate was ini-
tially selected as a model substrate. After the reaction, thin layer
chromatography analysis showed that linalyl acetate converted
to linalool by EstEH112 (Fig. S2). Therefore, this enzyme can effi-
ciently catalyze the hydrolysis of tertiary alcohol esters to tertiary
alcohol. Because many valuable tertiary alcohols contain of bulky
hydrophobic residues, the efficient and enantioselective hydrolysis
Xylene
a
log P value is the partition coefficient of an organic solvent between water and
n-octanol phases.
b
The enzyme solutions were incubated with organic solvents at various concen-
trations for 1 h at 35 ◦C with shaking at 1400 rpm. The residual activity of EstEH112
relative to non-solvent containing enzyme solution was measured. All assays were
performed three times under standard assay condition.

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K.-H. Oh et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 67–73
Table 2
tertiary alcohols. However, in most cases, GGG(A)X-motif hydro-
lases do not show high enantioselectivity [21,22]. It have been
reported that exchange of glycine in this motif to alanine and
structure based mutations of other amino acid positions in
substrate binding pocket influenced the activity and enantios-
electivity of esterase toward TAEs [32,33]. Therefore, although
the enantioselectivity of EstEH112 toward the investigated com-
Activity and enantioselectivity of EstEH112 toward tertiary alcohol acetates.a
Compound
a
b
c
d
e
f
gb
h
Conversion (%)
Enantioselectivity
59
3
55
1
53
4
50
3
47
3
n.c
41
4
n.c
n.c, no conversion. The enantioselectivity of the enzyme in the resolution of each
substrate was calculated by the formula: E = [ln[1 − c](1 + eep)]/[ln[1 − c](1 − eep)].
a
pounds is not satisfying, this can be
a good starting point
4-h samples.
24-h sample.
b
for further protein engineering studies of this esterase, espe-
cially when the structure of EstEH112 is resolved, to extend
the application of EstEH112 for the synthesis of enantio-pure
tertiary alcohols through structure based protein engineer-
ing.
of these ester forms is important. EstEH112 showed the catalytic
activity toward a wide range of bulky synthetic tertiary alcohol
acetates (Table 2). Most of tested TAEs were efficiently hydrolyzed
in 4 h, but enantioselectivities were low. Compounds f and h, how-
ever, are not converted by EstEH112. In previous studies, these
two compounds were also converted by only a limited number of
esterases [24,25].
Acknowledgements
The 21C Frontier Program of Microbial Genomics and Appli-
cations (Grant MG05-0401-2-0) from the Ministry of Education,
Science and Technology (MEST) of the Republic of Korea supported
this work. European Social Funds and VentureCup M-V (UG09005)
are also gratefully acknowledged for financial support. G.S.N. is
grateful to the DAAD (Grant: A/07/95194) and the Vietnamese
ministry of Education and Training (Grant: 3413/QDBGDDT-
VP).
4. Discussion
The metagenomic library from intertidal flat sediment was
screened for lipolytic enzymes based on functional screening.
Amino acid sequence analysis exhibited that the isolated EstEH112
contained the GGG(A)X-motif in the oxyanion hole, the typical con-
served sequence motifs of esterase/lipase, GXSXG, and a putative
catalytic triad composed of Ser185, Asp281, His311. EstEH112 pre-
ferred short-chain p-nitrophenylesters as substrate and showed
optimal activity at pH 8.0 and 35 ◦C. EstEH112 was stable up to
40 ◦C though it became inactivated rapidly at 55 ◦C. It displayed
approximately 30% of its activity at 10 ◦C compared to the level of
activity at its optimal temperature. These characteristics are sim-
ilar to other lipolytic enzymes isolated from metagenomic library
of marine environments.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.04.015.
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