characterization of a novel alkali ne family viii esterase with s ... · enantiomer, such as...
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February 2016⎪Vol. 26⎪No. 2
J. Microbiol. Biotechnol. (2016), 26(2), 315–325http://dx.doi.org/10.4014/jmb.1509.09081 Research Article jmbReviewCharacterization of a Novel Alkaline Family VIII Esterase with S-EnantiomerPreference from a Compost Metagenomic LibraryHyun Woo Lee1, Won Kyeong Jung1, Yong Ho Kim2, Bum Han Ryu3, T. Doohun Kim3 , Jungho Kim2, and
Hoon Kim1,2*
1Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic
of Korea2Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea3Department of Chemistry, Sookmyung Women's University, Seoul 04312, Republic of Korea
Introduction
Lipolytic enzymes, esterases and lipases, are carboxylic
ester hydrolases. Esterases (E.C. 3.1.1.1) hydrolyze ester
bonds of short fatty acids with less than 10 carbons, and
lipases (E.C. 3.1.1.3) hydrolyze ester bonds of long fatty
acids with more than 10 carbons. Esterases and lipases,
particularly with their enantioselectivity, have a wide range
of biological applications in the synthesis of biopolymers,
pharmaceuticals, agrochemicals, and flavor compounds
[12]. Bacterial lipolytic enzymes were originally grouped
into eight families [1] and the classification has been
expanded to 15 families with the recent discovery of many
new lipolytic enzymes [6]. Originally, three enzymes that
were approximately 380 residues long and showed striking
similarity to several class C β-lactamases were classified as
family VIII enzymes [1]. Although lipolytic family VIII
enzymes share common motifs such as GxxK with the β-
lactamase family, they are thought to be evolutionally
rather loosely related to other esterases [1, 30]. Very recently,
several family VIII esterases from diverse metagenomic
sources have been experimentally studied [3, 7, 21, 29].
The microbial community in compost includes myriad
microorganisms, and the indigenous microbes of compost
Received: September 30, 2015
Revised: October 22, 2015
Accepted: October 23, 2015
First published online
October 27, 2015
*Corresponding author
Phone: +82-61-750-3751;
Fax: +82-61-750-3708;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2016 by
The Korean Society for Microbiology
and Biotechnology
A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene
encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be
44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is
an esterase from a drinking water metagenome, when compared with the enzymes with
reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the
typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have
the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW,
PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of
the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be
classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at
40ºC and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30%
methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate,
and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates,
tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an S-
enantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance
to methanol and the substrate specificity may provide potential advantage in the use of the
enzyme in pharmaceutical and other biotechnological processes.
Keywords: Compost metagenomic library, enantioselectivity, family VIII esterase, methanol
activation, short-length p-nitrophenyl esters
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316 Lee et al.
J. Microbiol. Biotechnol.
produce various enzymes. The microbial community of
compost is expected to be more diverse than that of any
other environment, since it varies greatly depending on the
nature of raw materials and the progress of composting [8,
45]. Many compost microorganisms would not be cultivated
in laboratories, and metagenomic approaches could be
employed to exploit various valuable genes from compost
microorganisms that are not easily culturable or totally
unculturable. Biocatalysts have been isolated from uncultured
microorganisms using a metagenomic approach [37, 39].
We have previously reported a novel esterase, Est2K,
from a compost metagenomic library [15]. In this study, we
isolated another novel esterase gene, est7K, from the library
and examined some properties of Est7K. The classification
of the family VIII enzymes into subfamilies, VIII.1 and
VIII.2, was also discussed with the analysis of additional
motifs in the family VIII enzymes.
Materials and Methods
Selection of Esterase-Positive Clones
A compost metagenomic library was constructed using a
fosmid vector, and esterase-positive clones were identified with
their clear zones on the LB agar plates containing 1% glyceryl
tributyrate (Sigma) as the substrate [15]. YH-E7, one of the 18
esterase-positive clones obtained, was used in this study.
Sequence Analysis and Construction of Phylogenetic Tree
The nucleotide sequence of the plasmid in an esterase-positive
clone was determined by SolGent (Daejeon, Korea). The conserved
region of the esterase was analyzed by BLASTp of NCBI (http://
www.ncbi.nlm.nih.gov), and the signal peptide was predicted by
SignalP 4.1 in CBS (http://www.cbs.dtu.dk/services/SignalP/) [33].
The molecular mass and pI of the encoded protein were analyzed
via the ExPASy site (http://www.expasy.ch/tools/protparam.html).
To construct a phylogenetic tree, the evolutionary history was
inferred using the neighbor-joining method [38]. Multiple alignments
of the amino acid sequences were performed using Clustal W [44]
and analyzed with GeneDoc 2.7 [25]. Evolutionary analyses were
conducted using the MEGA6 program [43].
Site-Directed Mutagenesis
Site-directed mutagenesis of the gene was conducted using a
QuikChange II kit (Stratagene, Santa Clara, CA, USA) [22]. The
primer pair for S73A mutation was 5’-CCGGATTGCTGCCAT
GACCAA-3’ (forward) and 5’-TTGGTCATGGCAGCAATCCGG-
3’ (reverse). The primer pair for Y191A mutation was 5’-
CGCAGTGGAATGCCTCTGTTTCAA-3’ (forward) and 5’-TTG
AAACAGAGGCATTCCACTGCG-3’ (reverse). The primer pair for
S356A mutation was 5’-TCATGCCCGCGGCCAAGGGCGAAT-3’
(forward) and 5’- ATTCGCCCTTGGCCGCGGGCATGA-3’ (reverse).
After 15 cycles, the PCR products were treated with DpnI, and the
resulting products were transformed to E. coli XL-1 blue super-
competent cells. The substitutions were confirmed by nucleotide
sequencing.
Determination of Esterase Activity
Esterase activity was determined by measuring the amount of
p-nitrophenol generated from p-nitrophenyl butyrate (Sigma), as
described previously [15]. Unless otherwise stated, the reaction
was performed for 1 min at 25ºC with 1 mM p-nitrophenyl
butyrate in 50 mM Tris-HCl (pH 8.0) and the absorbance at
400 nm of the reaction mixture was continuously measured. One
unit of esterase activity was defined as the amount of enzyme that
generated 1 µmol of p-nitrophenol in 1 min under the conditions.
Purification of the Enzyme
Crude enzyme preparation and purification of the enzyme were
performed as previously described [15, 42]. Briefly, the transformants
were grown in LB broth containing ampicillin for 12 h at 37ºC,
harvested, and dispersed in 50 mM sodium citrate (pH 5.5), and
dialyzed against 50 mM Tris-HCl (pH 8.0) buffer. The enzyme was
purified by High-Q (5 ml; Bio-Rad, CA, USA), CHT-II (5 ml; Bio-
Rad), and t-Butyl HIC (5 ml; Bio-Rad) column chromatographies.
The progress of the purification was monitored by determining the
amount of protein [19] and SDS-PAGE on an 11.5% polyacrylamide
gel [18].
Characterization of the Enzyme
The optimum temperature, thermostability, and optimum pH
of the enzyme activity were determined as described previously
[15]. The influences of cations and phenylmethylsulfonyl fluoride
(PMSF), substrate specificity of the enzyme, and hydrolysis of the
β-lactam antibiotic ampicillin were determined as described
previously [15].
Enantioselectivity was analyzed using a pH shift assay; Est7K
was reacted with 300 mM (R)- or (S)-methyl-3-hydroxy-2-
methylpropionate in 20 mM Tris-HCl (pH 8.0) containing phenol
red (2 g/l) [14]. The absorbance spectra of the solutions were
recorded at 350–600 nm. For the hydrolysis of tertiary alcohol
esters, Est7K was reacted with 25 mM t-butyl acetate, linalyl acetate,
or α-terpinyl acetate in 20 mM Tris-HCl, pH 8.0, containing
phenol red [24]. Hydrolysis of glyceryl esters (glyceryl butyrate
and glyceryl trioleate) and oils (fish oil and olive oil) were
analyzed with 1% substrates.
Nucleotide Sequence Accession Number
The nucleotide sequence of the esterase gene est7K has been
deposited at GenBank under the accession number KP756684.
Results
Characterization of Esterase Gene est7K and Est7K
In the previous study, 18 esterase-positive subclones
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February 2016⎪Vol. 26⎪No. 2
were obtained from the mixed DNA of the 19 active fosmid
clones of a compost metagenome, and a novel esterase
gene, est2K, was isolated and characterized from one of the
subclones [15]. In this study, the other 17 subclones were
tested for their lipolytic activity and 13 were found to be
positive and four were very weakly positive. The sequences
of the 13 putative lipolytic genes from the positive clones
were determined and eight different lipolytic genes were
identified (data not shown). The lipolytic gene of subclone
YH-E7 showed the lowest similarity to the reported
lipolytic genes and was selected for further study.
The recombinant plasmid of YH-E7 had an inserted DNA
fragment of about 2.4 kb. Sequence analysis of the inserted
DNA fragment revealed an ORF of 1,236 bp, and the ORF
was identified to be an esterase gene with a domain that
showed high similarity to the β-lactamase superfamily and
was named est7K. The esterase gene est7K was expected to
encode a protein of 411 amino acid residues with no signal
peptide. Est7K was calculated to be an acidic protein of
44,969 Da with a theoretical pI of 5.89.
The amino acid sequence of Est7K showed the highest
identity of 74% to that of β-lactamase of Citromicrobium sp.
JLT1363 (WP_033193084), followed by 74% identity to the
β-lactamase of C. bathyomarinum (WP_010239180), 71%
identity to the β-lactamase of uncultured Sphingomonadales
bacterium HF0500_24B12 (ADI19464), and 66% identity to
the β-lactamase of Erythrobacter litoralis (WP_011413033).
The above sequences have been deposited very recently
and their enzymatic properties have not yet been reported.
Among the family VIII lipolytic enzymes with reported
enzymatic properties, EstA3 from drinking water metagenome
[9] and EstF4K from soil/water metagenome [29] showed
the highest identity of 57% to Est7K.
Analysis of the amino acid sequences of Est7K and 23
lipolytic family VIII enzymes revealed that Est7K contained
conserved regions such as SMTK (73rd to 76th), YSV (191st to
193rd), and WGG (362nd to 364th) motifs (Fig. 1).
The alignment suggests that, in addition to the three
motifs mentioned above, three more motifs are conserved
in most of the family VIII enzymes: LxxxPGxxW (181st to
189th) at the N-terminus of the YSV motif, PLGMxDTxF
(221st to 229th), and GGxG (271st to 274th) (Fig. 1). The amino
acid residues Gly54, Pro103, and Gly140, which can form β-
turns in the 3D structure of the enzymes, are observed in
all the family VIII enzymes (Fig. 1). Asp281 and Gly358 are
conserved in the 19 enzymes of family VIII, but are
replaced with Gly and Arg, respectively, in the five family
VIII enzymes (Fig. 1). The GxSxG motif was found to be not
conserved in all the family VIII enzymes. Only two
enzymes, EstC and Est2K, have the GxSxG sequence; nine
enzymes, including Est7K, have xxSxG (354th to 358th), and
the other enzymes do not have the sequence at all (Fig. 1).
In the phylogenetic tree of Est7K and other related
proteins, Est7K was clustered with family VIII esterases
and the location of Est7K in the tree suggested Est7K to be
a novel member of the family (Fig. 2). It was also suggested
from the tree that the family VIII enzymes could be divided
into two subfamilies (Fig. 2).
Site-Directed Mutagenesis
Site-directed mutagenesis of Ser73 to Ala73 resulted in
complete loss of enzyme activity, and that of Tyr191 to
Ala191 caused almost complete loss (93.4%) of the activity.
The mutagenesis of Ser356 to Ala356 did not cause any
significant change in the enzyme activity (data not shown).
Purification of Est7K
Est7K produced by the clone YH-E7 was purified by
High-Q, CHT-II, and t-butyl HIC chromatography. The
recovery rate, purification fold, and specific activity of the
enzyme were 15.2%, 67.5-fold, and 790.2 U/mg protein,
respectively (data not shown). The specific activity of
Est7K was 46.2 times higher than that of Est2K (17.1 U/mg
protein) under the same assay conditions [15]. Purified
Est7K appeared as a single band on an SDS-PAGE gel and
had a molecular mass of 42.4 kDa (Fig. 3).
Properties of Est7K
Est7K exerted its maximal activity at 40ºC, and showed
80% of the maximal activity at 50ºC (Fig. 4A). Est7K
retained 65% of the original activity after 30 min of heat
treatment at 30ºC, but was completely inactivated after
15 min at 40ºC (Fig. 4B). Est7K showed its maximal activity
at pH 10 (Fig. 4C). Mono- and divalent cations showed no
significant influence on the enzyme activity, except that
5 mM Cu2+ inhibited 53.9% of the activity (Table 1). PMSF
at a concentration of 1 mM inhibited 62.4% of the activity
(Table 1), thereby indicating that serine residue is responsible
for the catalytic activity. Est7K was stable in the presence of
polar organic solvents methanol and isopropanol, but was
sensitive to acetonitrile, losing more than 65% of the
activity in the presence of 5% and 30% acetonitrile (Table 1).
The increase in the concentration of methanol from 5% to
30% resulted in a significant increase in the enzyme
activity, from 107.5% to 208.5% (Table 1). No such effect
was observed with isopropanol.
When pNP esters were used as substrates, purified Est7K
efficiently hydrolyzed ester bonds of short-chain fatty
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318 Lee et al.
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Fig. 1. Alignment of the amino acid sequences of Est7K (Accession No. KP756684) and related lipolytic enzymes. Sequences: esterases EstA3 and EstCE1 from drinking water and soil metagenomes (DQ022078 and DQ022079, respectively), putative β-lactamase
class C EstF4K from soil and water metagenomes (JN001202), hypothetical protein Est22 (shown as Est22-S in this figure) of a soil metagenome
(HQ156921), lipolytic enzyme LipBL from Marinobacter lipolyticus SM19 (FR719924), esterases EstM-N1 and EstM-N2 from an arctic soil
metagenome (HQ154132 and HQ154133, respectively), EstC of a soil metagenome (FJ025785), Est2K from a compost metagenome (GQ426329),
EstU1 from a soil metagenome (JF791800), Est22 from a leachate metagenome (KF052088), SBLip1 from a forest soil metagenome (JQ780827), Est01
from a biogas slurry metagenome (HQ444406), EstB from Burkholderia gladioli (AF123455), Lpc53E1 from a marine sponge metagenome (JQ659262),
PBS-2 from Paenibacillus sp. PBS-2 (KF972440), EstBL from Burkholderia multivorans UWC10 (AAX78516), Lip8 from Pseudomonas aeruginosa LST-03
(AB126049), esterase III (Est III) from Pseudomonas fluorescens SIK WI (AAC60471), EstA (EstA-Sc) from Streptomyces chrysomallus X2 (CAA78842),
EstA (EstA-An) from Arthrobacter nitroguajacolicus Rü61a (CAD61039), EstA (EstA-P) from Pseudomonas sp. LS107d2 (M68491), and Est from
Arthrobacter globiformis SC-6-98-28 (AAA99492). Consensus sequences are shown with uppercase or lowercase letters. Number 6 represents the
branched hydrophobic amino acid residues Val/Leu/Ile. The conserved motifs are boxed in yellow and newly identified motifs are boxed in red.
The region with the xxSxG sequence is shown in a blue box.
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acids, and the relative activities toward pNP-butyrate (C4),
pNP-acetate (C2), pNP-octanoate (C8), and pNP-caprate
(C10) were 100%, 59.3%, 11.4%, and 3.5%, respectively
(Table 2). The ratio of Est7K activity for pNP-C10 to pNP-
C4 was very low (0.035). The value was similar to those of
some family VIII esterases, EstCE1 (0.03) and EstA3 (0) [9],
but was lower than those of other family VIII esterases,
Est2K (0.4) [15] and Est22 (0.3) [21].
The Km values for pNP-C4, pNP-C2, pNP-C8, and pNP-
C10 were 61.7, 123.9, 86.6, and 25.0 µM, respectively
(Table 2). Est7K showed no or negligible β-lactamase activity
toward ampicillin and nitrocefin under the experimental
conditions, even though Est7K was grouped as the same
family VIII with class C β-lactamases (data not shown).
Est7K hydrolyzed an S-enantiomer, (S)-methyl-3-hydroxy-
2-methylpropionate, more efficiently than an R-enantiomer
(Fig. 5A). The conversion ratio of R:S was calculated to be
1:2.53 by comparing A560 after 10 min reaction (Fig. 5B).
Est7K could not hydrolyze the tertiary alcohols such as t-
butyl acetate, linalyl acetate, or α-terpinyl acetate (data not
shown). Furthermore, Est7K efficiently hydrolyzed glyceryl
tributyrate, but did not hydrolyze glyceryl trioleate, fish
oil, and olive oil (Fig. 5C).
Discussion
Est7K showed high identities to the recently reported
β-lactamases of unreported enzymatic properties. Of
approximately 100 listed sequences at NCBI BLAST that
Fig. 2. Phylogenetic tree showing the evolutionary relationships and levels of homology of the lipolytic enzymes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The
analysis involved 24 amino acid sequences. Evolutionary analyses were conducted in MEGA6 [43].
Fig. 3. SDS-PAGE of the purified Est7K. M, molecular weight markers; lane 1, crude extract of the clone; lane 2,
pooled from High-Q chromatography; lane 3, pooled from CHT-II
chromatography; lane 4, pooled from HIC chromatography.
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320 Lee et al.
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shows over 49% identity with Query coverage of 97%, only
a few sequences have been published with information on
their enzymatic properties; EstA3 from drinking water
metagenome, EstCE1 from soil metagenome [9], EstF4K
from soil/water metagenome [29], and Est22 from soil
metagenome [23]. Recently, enzymatic properties of
several lipolytic family VIII enzymes have been reported:
Est22 from leachate metagenome [21], SBLip1 from forest
soil metagenome [3], Est01 from biogas slurry metagenome
[7], and PBS-2 from Paenibacillus sp. PBS-2 [16].
The SMTK motif in Est7K corresponds to the SxxK motif
found in most of the β-lactamase superfamily [1, 46]. The
YSV and WGG motifs of Est7K correspond to the YSL (or
YXN) and WGG (or KTG box) motifs, respectively, that are
conserved in family VIII enzymes [46]. It has been
suggested that the Ser in the SxxK motif functions as a
nucleophile, and the Tyr in the YSL motif activates the Ser
as a general base for attacking the ester-carbonyl of the
substrate molecule and is stabilized by the proximity of the
side chains of Lys in the SxxK motif and Trp in the WGG
motif [36, 46]. The loss of enzyme activity due to the site-
Table 1. Effects of cations, an inhibitor, and organic solvents onthe activity of Est7K.
Relative activity (%)a
Control (+ 1% Isopropanol) 100.0
Cation (5 mM)
K+ 94.6 ± 3.5
Na+ 109.7 ± 7.8
Ca2+ 108.5 ± 5.3
Mg2+ 75.2 ± 3.7
Mn2+ 86.5 ± 3.2
Zn2+ 79.9 ± 2.7
Cu2+ 46.1 ± 3.3
Inhibitor (1 mM)
PMSF 37.6 ± 8.7
Solvent
Isopropanol (5%) 78.0 ± 1.4
Isopropanol (30%) 106.5 ± 3.7
Methanol (5%) 107.5 ± 6.2
Methanol (30%) 208.5 ± 3.2
Acetonitrile (5%) 27.8 ± 8.8
Acetonitrile (30%) 33.7 ± 8.9
aRelative activity was expressed as specific activities relative to the activity
790.2 U/mg protein. The values represent the average of the results from
independent triplicate experiments.
Fig. 4. Effects of temperature and pH on the enzyme activity ofEst7K.
(A) Optimum temperature; (B) thermostability; and (C) optimum pH.
Enzyme activities were measured by a continuous method at each
designated pH. For more details, refer to the Materials and Methods
section.
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directed mutagenesis of Ser73 to Ala73 and of Tyr191 to
Ala191 indicates that the Ser in the SMTK motif and Tyr in
the YSV motif are involved in catalytic function. The
conserved residue Gly140 is positioned at the end of the
motif LLxHxxG, which was described as a family VIII
motif [32, 34]. However, the motif is not highly conserved,
except that the Gly is absolutely conserved and the first
residue is moderately conserved as branched hydrophobic
amino acids Leu/Val/Ile.
The typical pentamotif GxSxG has been reported to
contain a serine residue that has a catalytic function in
most esterase families [4]. The GxSxG sequence was found
Table 2. Substrate specificity of Est7K on pNP-esters.
Specific activity (U/mg) Relative activity (%) Km (µM)
PNP-acetate (C2) 468.3 59.3 61.7
PNP-butyrate (C4) 790.2 100.0 123.9
PNP-octanoate (C8) 89.8 11.4 86.6
PNP-caprate (10) 27.8 3.5 25
PNP-laurate (12) 6.8 0.9 ND
PNP-myristate (14) 4.4 0.6 ND
PNP-palmitate (16) 4.4 0.6 ND
ND, not determined.
Fig. 5. Analysis of enantioselectivity and lipid hydrolysis of Est7K using pH shift assay. (A) Enantioselectivity analysis with (R)- or (S)-methyl-3-hydroxy-2-methylpropionate containing phenol red: 1, (R) substrate; 2, (R) substrate +
enzyme; 3, (S) substrate; 4, (S) substrate + enzyme. (B) Absorbance spectra of the reaction mixtures in (A). (C) Hydrolysis of glyceryl esters and
oils.
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not to be conserved in Est7K as in most of the family VIII
enzymes aligned. The pentamotif sequence was reported to
be only partially conserved when a family VIII esterase,
EstA from Arthrobacter nitroguajacolicus, two other family
VIII esterases, and AmpC were aligned [40]. No loss of the
enzyme activity by the site-directed mutagenesis of Ser356
in Est7K to Ala356 indicates that the Ser residue in the
xxSxG sequence has no important catalytic function,
although Est7K has the xxSxG (354th to 358th) sequence. The
motifs GMS372RG of Est2K, GMS373EG of EstC, GIS149DG of
EstB, and GLS321VG of LipBL were reported to be the
GxSxG motif, but the Ser in the motifs was found not to be
essential for the catalytic activity [15, 30-32, 36]. The role of
the nucleophilic Ser residue in the SxxK motif in the family
VIII enzymes is thought to be the same as that of the Ser
residue in the GxSxG motif in most of other lipolytic
enzymes and in the GDSL motif in family II (GDSL family).
Based on the above results, it could be concluded that the
catalytic residues in the active sites of the family VIII esterases
are Ser, Lys, and Tyr, whereas those of the superfamily of
α/β-hydrolases, including lipolytic enzymes, are Ser, Asp,
and His, which form a catalytic triad [1, 28].
In the phylogenetic tree of family VIII enzymes, five
enzymes that showed identities lower than 25% to Est7K
were clustered together and were clearly separated from
other enzymes (Fig. 2). The five enzymes were Est from
A. globiformis [26], EstA from A. nitroguajacolicus [40], EstA
from Streptomyces chrysomallus [2], esterase III from
Pseudomonas fluorescens [17], and EstA from Pseudomonas
sp. LS107d2 [20]. Interestingly, the alignment analysis
showed that the conserved residues in the six motifs of the
five enzymes were different from those of the other 19
enzymes: (i) the second residue in SxxK of the five enzymes
is C (M/Vin the 19 enzymes); ii) the second and third
residues in YSV is H/E and A, respectively; iii) the first
residue in WGG is H; iv) the first and the last residues in
LxxxPGxxW are P and H/F, respectively; v) the last
residue in PLGMxDTxF is V/L; and vi) the first and the last
residues in GGxG are W/S and A/M, respectively (Table 1).
With these results, it is suggested that family VIII be
classified into two subfamilies, VIII.1 and VIII.2, and that
subfamily VIII.1 include the reference enzyme Est from
A. globiformis (AAA99492), as in the first classification of
family VIII by Argipny and Jaeger [1]. Family I lipolytic
enzymes have already been divided into six subfamilies
[1].
Enzymes belonging to lipolytic family VIII are listed with
some of their characteristics in order, based on their
identities to Est7K, in Table 3. Est7K had no signal peptide
like most of the lipolytic family VIII enzymes reported. To
date, only five lipolytic family VIII enzymes have been
reported to have a signal peptide: EstC [36], Est2K [15],
EstU1 [13], Est22 [21], and SBLip1 [3]. The five enzymes
were derived from metagenomic sources and showed
identities of 34–40% to Est7K (Table 3).
Est7K was a mid-sized enzyme with 411 amino acid
residues among the family VIII enzymes that had 378 to 445
amino acid residues (Table 3). The optimum temperature
(40ºC) of Est7K was in the middle range compared with
those of other family VIII enzymes (20–80ºC) and the
optimum pH of Est7K was in the alkaline region, pH 10.0
(Table 3).
Est7K was tolerant to methanol and a more than 2-fold
increase in the activity was observed in the presence of 30%
methanol. A similar effect of methanol was reported with
EstF4K (175.8% at 30% methanol) and Lpc53E1 (220% at
20% methanol), and a more striking effect with EstC (600%
at 20% methanol) (Table 3).
Est7K, like most of the family VIII enzymes, preferred a
short-chain fatty acid (C4) as the substrate (Table 3). Only
Lpc53E1 preferred a long-chain fatty acid (C16) as the
substrate [41]. The substrate preference indicates that
Est7K is a typical carboxylesterase rather than a lipase [1].
The substrate specificity of Est7K might have been changed
during the evolutionary process, as it has been suggested
for the family VIII esterases that did not show β-lactamase
activity due to steric reasons [46] or the length of the Ω-
loop [5]. Est7K showed moderate to low levels of
enantioselectivity for (S)-enantiomer of methyl-3-hydroxy-
2-methylpropionate. Although EstCE1, which has low
identity to Est7K, was highly enantioselective for (+)-
menthyl acetate [9], the enzymes with high identity to
Est7K, EstA3, EstF4K, and LipBL showed relatively low
levels of enantioselectivity [9, 29, 31] (Table 3).
Est7K did not hydrolyze tertiary alcohol esters. Only
EstC and EstB have been reported to hydrolyze the esters
of tertiary alcohol [36, 46] (Table 3). No other enzymes have
been studied for their ability to hydrolyze tertiary alcohols
(Table 3). The GGGX motif, which is located in the active
site adjacent to the oxyanion, is linked with the hydrolysis
of esters of tertiary alcohols [10, 36]. Although the GGGL
sequence in EstC, corresponding to the GGGX motif, was
found to be located toward the C-terminus of the enzyme,
the ability of EstC to hydrolyze linalyl acetate was suggested
to support the importance of this motif [36]. Many of the
family VIII enzymes, including Est7K, have the GGGL
sequence at the corresponding position of EstC (Fig. 1).
However, Est7K could not hydrolyze the tertiary alcohol,
-
An
Alkalin
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III Esterase from
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Table 3. Comparison of enzyme properties of Est7K and other bacterial family VIII lipolytic enzymes.a
Enzyme SourceIdentity
(%)Signal
peptide
Amino acid
residues
Optimum temperature
(°C)
Optimum pH
Preferred pNP
estersb
Isopropanolc
(30%)Methanolc
(30%)Acetonitrilec
(30%)β-Lactamase
activityEnantio-
selectivity
Tertiary alcohol esters
Reference
Est7K Compost metagenome 100 No 411 40 10.0 C4 106.5 208.5 33.7 - + - This study
EstA3 Drinking water metagenome 57 396 50 9.0 C4 117 130 87 - + [9]
EstF4K Soil/water metagenome 57 396 50 8.0 C3, C4 175.8 16.5 - + [29]
Est22 Soil metagenome 49 424 C4 [23]
LipBL Marinobacter lipolyticus SM19 49 404 80 7.0 C6 98.4 120.5 27.7 + [31]
EstM-N2 Arctic soil metagenome 46 407 30 9.0 C4 L [47]
EstC Leachate metagenome 40 Yes 427 40 C4 ~100 ~600 20 L + [36]
Est2K Compost metagenome 38 Yes 432 50 10.0 C4 23.5 93.7 42.2 - [15]
EstU1 Soil metagenome 37 Yes 426 45 8.5 C4 + [13]
Est22 Leachate metagenome 35 Yes 423 30 8.0 C4 + [21]
SBLip1 Forest soil metagenome 34 Yes 445 35 10.0 C4 ~2(at 20%)
~60(at 20%)
~0(at 20%)
L [3]
Est01 Biogas slurry metagenome 33 397 20 8.0 C4 - [7]
EstM-N1 Arctic soil metagenome 33 395 20 9.0 C4 L [47]
EstB Burkholderia gladioli 31 392 43 7.0 C4 -f + [32]
Lpc53E1 Marine sponge metagenome 31 387 40 7.0 C16 176.4(at 20%)
220.5(at 20%)
271.2(at 20%)
- [41]
PBS-2 Paenibacillus sp. PBS-2 30 No 377 30 9.0 C4 104 102 62 + [16]
EstBL Burkholderia multivorans UWC10
30 398 C2 - [35]
Lip8 Pseudomonas aeruginosa LST-03
30 No 391 30 7.0 C2e ~0(at 25%)
[27]
EstCE1 Soil metagenome 27 388 47 10.0 C4 23 0 0 - ++ [9]
EstIII Pseudomonas fluorescens SIK WI 25 (46)d 382 50 9.5 C2 [17]
EstA Streptomyces chrysomallus X2 24 (71) 389 C4 [2]
EstA Arthrobacter nitroguajacolicus Rü61a
23 (63) 372 50~60 9.5 C2, C4 115 120 [40]
EstA Pseudomonas sp. LS107d2 23 (47) 389 C4 [20]
Est Arthrobacter globiformis SC-6-98-28
23 (44) 375 +g [26]
aEnzymes are listed in order based on their identity to Est7K and availability of experimental data. bp-Nitrophenyl esters; crelative activities (%) at the concentration of solvents. dNumerals in parentheses represent query coverage; eTriacetin; fEster bond, not β-lactam ring, of 7-amino cephalosporinic acid was
cleaved; gstereoselectivity for production of (+)-trans-chrysanthemic acid.
-, Negative activity; +, positive activity; ++, high activity; L, low activity. Blank, not available.
-
324 Lee et al.
J. Microbiol. Biotechnol.
and hydrolysis by other enzymes has not yet been reported
(Table 3). EstB hydrolyzing the tertiary alcohol has a
GAGM sequence, but not GGGX. It is likely that the GGGL
sequence in Est7K and EstC is not responsible for the
hydrolysis of the tertiary alcohol.
In this study, we characterized a new family VIII
esterase, Est7K, without the GxSxG motif found in most of
the β-lactamase superfamily. The family VIII enzymes were
found to have three additional motifs conserved and
suggested to be classified into two subfamilies, VIII.1 and
VIII.2. The tolerance to methanol and the enantioselectivity
of Est7K may provide potential advantage in the use of the
enzyme in pharmaceutical and other biotechnological
processes.
Acknowledgments
This study was supported by the 21C Frontier Microbial
Genomics and Application Center Program, Ministry of
Science, ICT and Future Planning, and by the Suncheon
Research Center for Natural Medicines, Korea.
References
1. Arpigny JL, Jaeger KE. 1999. Bacterial lipolytic enzymes:
classification and properties. Biochem. J. 343: 177-183.
2. Berger R, Hoffmann M, Keller U. 1998. Molecular analysis
of a gene encoding a cell-bound esterase from Streptomyces
chrysomallus. J. Bacteriol. 180: 6396-6399.
3. Biver S, Vandenbol M. 2013. Characterization of three new
carboxylic ester hydrolases isolated by functional screening
of a forest soil metagenomic library. J. Ind. Microbiol.
Biotechnol. 40: 191-200.
4. Bornscheuer UT. 2002. Microbial carboxylesterase: classification,
properties and application in biocatalysis. FEMS Microbiol.
Rev. 26: 73-81.
5. Cha SS, An YJ, Jeong CS, Kim MK, Jeon JH, Lee CM, et al.
2013. Structural basis for the β-lactamase activity of EstU1, a
family VIII carboxylesterase. Proteins 81: 2045-2051.
6. Charbonneau DM, Beauregard M. 2013. Role of key salt
bridges in thermostability of G. thermodenitrificans EstGtA2:
distinctive patterns within the new bacterial lipolytic enzyme
family XV. PLoS One 8: e76675.
7. Cheng X, Wang X, Qiu T, Yuan M, Sun J, Gao J. 2014.
Molecular cloning and characterization of a novel cold-adapted
family VIII esterase from a biogas slurry metagenomic
library. J. Microbiol. Biotechnol. 24: 1484-1489.
8. Cho KM, Lee SM, Math RK, Islam SM, Kambiranda DM,
Kim JM, et al. 2008. Culture-independent analysis of
microbial succession during composting of swine slurry and
mushroom cultural wastes. J. Microbiol. Biotechnol. 18: 1874-
1883.
9. Elend C, Schmeisser C, Leggewie C, Babiak P, Carballeira
JD, Steele HL, et al. 2006. Isolation and biochemical
characterization of two novel metagenome-derived esterases.
Appl. Environ. Microbiol. 72: 3637-3645.
10. Henke E, Bornscheuer UT, Schmid RD, Pleiss J. 2003. A
molecular mechanism of enantiorecognition of tertiary
alcohols by carboxylesterases. Chembiochem 4: 485-493.
11. Jaeger KE, Dijkstra BW, Reetz MT. 1999. Bacterial
biocatalysis: molecular biology, three-dimensional structures,
and biotechnological applications of lipases. Annu. Rev.
Microbiol. 53: 315-351.
12. Jagger KE, Eggert T. 2002. Lipases for biotechnology. Curr.
Opin. Biotechnol. 13: 390-397.
13. Jeon JH, Kim SJ, Lee HS, Cha SS, Lee JH, Yoon SH, et al.
2011. Novel metagenome-derived carboxylesterase that
hydrolyzes β-lactam antibiotics. Appl. Environ. Microbiol. 77:
7830-7836.
14. Kim HJ, Jung WK, Lee HW, Yoo W, Kim TD, Kim H. 2015.
Characterization of an alkaline family I.4 lipase from Bacillus
sp. W130-35 isolated from a tidal mud flat with broad
substrate specificity. J. Microbiol. Biotechnol. DOI: 10.4014/
jmb.1507.07104 [In Press].
15. Kim YH, Kwon EJ, Kim SK, Jeong YS, Kim J, Yun HD, Kim
H. 2010. Molecular cloning and characterization of a novel
family VIII alkaline esterase from a compost metagenomic
library. Biochem. Biophys. Res. Commun. 393: 45-49.
16. Kim YO, Park IS, Nam BH, Kim DG, Jee YJ, Lee SJ, An CM.
2014. A novel esterase from Paenibacillus sp. PBS-2 is a new
member of the β-lactamase belonging to the family VIII
lipases/esterases. J. Microbiol. Biotechnol. 24: 1260-1268.
17. Kim YS, Lee HB, Choi KD, Park S, Yoo OJ. 1994. Cloning of
Pseudomonas fluorescens carboxylesterase gene and characterization
of its product expressed in Escherichia coli. Biosci. Biotechnol.
Biochem. 58: 111-116.
18. Laemmli UK. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227:
680-685.
19. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. 1951.
Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193: 265-275.
20. McKay DB, Jennings MP, Godfrey EA, MacRae IC, Rogers
PJ, Beacham IR. 1992. Molecular analysis of an esterase-
encoding gene from a lipolytic psychrotrophic pseudomonad.
J. Gen. Microbiol. 138: 701-708.
21. Mokoena N, Mathiba K, Tsekoa T, Steenkamp P, Rashamuse K.
2013. Functional characterisation of a metagenome derived
family VIII esterase with a deacetylation activity on β-lactam
antibiotics. Biochem. Biophys. Res. Commun. 437: 342-348.
22. Na HB, Jung WK, Jeong YS, Kim HJ, Kim SK, Kim J, et al.
2015. Characterization of a GH family 8 b-1,3-1,4-glucanase
with distinctive broad substrate specificity from Paenibacillus
sp. X4. Biotechnol. Lett. 37: 643-655.
-
An Alkaline Family VIII Esterase from a Compost Metagenomic Library 325
February 2016⎪Vol. 26⎪No. 2
23. Nacke H, Will C, Herzog S, Nowka B, Engelhaupt M,
Daniel R. 2011. Identification of novel lipolytic genes and
gene families by screening of metagenomic libraries derived
from soil samples of the German Biodiversity Exploratories.
FEMS Microbiol. Ecol. 78: 188-201.
24. Ngo TD, Ryu BH, Ju H, Jang EJ, Kim KK, Kim TD. 2014.
Crystallographic analysis and biochemical applications of a
novel penicillin-binding protein/β-lactamase homologue from
a metagenomic library. Acta Crystallogr. D Biol. Crystallogr.
70: 2455-2466.
25. Nicholas KB, Nicholas Jr HB. 1997. GeneDoc: a tool for
editing and annotating multiple sequence alignments.
Distributed by the authors.
26. Nishizawa M, Shimizu M, Ohkawa H, Kanaoka M. 1995.
Stereoselective production of (+)-trans-chrysanthemic acid
by a microbial esterase: cloning, nucleotide sequence, and
overexpression of the esterase gene of Arthrobacter globiformis
in Escherichia coli. Appl. Environ. Microbiol. 61: 3208-3215.
27. Ogino H, Mimitsuka T, Muto T, Matsumura M, Yasuda M,
Ishimi K, Ishikawa H. 2004. Cloning, expression, and
characterization of a lipolytic enzyme gene (lip8) from
Pseudomonas aeruginosa LST-03. J. Mol. Microbiol. Biotechnol.
7: 212-223.
28. Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken
SM, et al. 1992. The alpha/beta hydrolase fold. Protein Eng.
5: 197-211.
29. Ouyang LM, Liu JY, Qiao M, Xu JH. 2013. Isolation and
biochemical characterization of two novel metagenome-
derived esterases. Appl. Biochem. Biotechnol. 169: 15-28.
30. Pérez D, Kovacic F, Wilhelm S, Jaeger KE, García MT,
Ventosa A, Mellado E. 2012. Identification of amino acids
involved in the hydrolytic activity of lipase LipBL from
Marinobacter lipolyticus. Microbiology 158: 2192-2203.
31. Pérez D, Martín S, Fernández-Lorente G, Filice M, Guisán
JM, Ventosa A, et al. 2011. A novel halophilic lipase, LipBL,
showing high efficiency in the production of eicosapentaenoic
acid (EPA). PLoS One 6: e23325.
32. Petersen EI, Valinger G, Solkner B, Stubenrauch G, Schwab
H. 2001. A novel esterase from Burkholderia gladioli shows
high deacetylation activity on cephalosporins is related to β-
lactamases and DD-peptidases. J. Biotechnol. 89: 11-25.
33. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011
SignalP 4.0: discriminating signal peptides from transmembrane
regions. Nat. Methods 8: 785-786.
34. Ranjan R, Grover A, Kapardar RK, Sharma R. 2005. Isolation
of novel lipolytic genes from uncultured bacteria of pond
water. Biochem. Biophys. Res. Commun. 335: 57-65.
35. Rashamuse KJ, Burton SG, Stafford WH, Cowan DA. 2007.
Molecular characterization of a novel family VIII esterase
from Burkholderia multivorans UWC10. J. Mol. Microbiol.
Biotechnol. 13: 181-188.
36. Rashamuse K, Magomani V, Ronneburg T, Brady D. 2009. A
novel family VIII carboxylesterase derived from a leachate
metagenome library exhibits promiscuous β-lactamase activity
on nitrocefin. Appl. Microbiol. Biotechnol. 83: 491-500.
37. Rondon MR, August PR, Bettermann AD, Brady SF, Grossman
TH, Liles MR, et al. 2000. Cloning the soil metagenome: a
strategy for accessing the genetic and functional diversity of
uncultured microorganisms. Appl. Environ. Microbiol. 66:
2541-2547.
38. Saitou N, Nei M. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol.
Evol. 4: 406-425.
39. Schmeisser C, Steele H, Streit WR. 2007. Metagenomics,
biotechnology with non-culturable microbes. Appl. Microbiol.
Biotechnol. 75: 955-962.
40. Schütte M, Fetzner S. 2007. EstA from Arthrobacter nitroguajacolicus
Rü61a, a thermo- and solvent-tolerant carboxylesterase related
class C β-lactamases. Curr. Microbiol. 54: 230-236.
41. Selvin J, Kennedy J, Lejon DP, Kiran GS, Dobson AD. 2012.
Isolation identification and biochemical characterization of a
novel halo-tolerant lipase from the metagenome of the
marine sponge Haliclona simulans. Microb. Cell Fact. 11: 72.
42. Shin ES, Yang MJ, Jung KH, Kwon EJ, Jung JS, Park SK, et
al. 2002. Influence of the transposition of the thermostabilizing
domain of Clostridium thermocellum xylanase (XynX) on
xylan binding and thermostabilization. Appl. Environ. Microbiol.
68: 3496-3501.
43. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013.
MEGA6: molecular evolutionary genetics analysis version
6.0. Mol. Biol. Evol. 30: 2725-2729.
44. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic Acids Res.
22: 4673-4680.
45. Tiquia SM. 2002. Evolution of extracellular enzyme activities
during manure composting. J. Appl. Microbiol. 92: 764-775.
46. Wagner UG, Petersen EI, Schwab H, Kratky C. 2002. EstB
from Burkholderia gladioli: a novel esterase with a β-
lactamase fold reveals steric factors to discriminate between
esterolytic and β-lactam cleaving activity. Protein Sci. 11:
467-478.
47. Yu EY, Kwon MA, Lee M, Oh JY, Choi JE, Lee JY, et al.
2011. Isolation and characterization of cold-active family VIII
esterases from an arctic soil metagenome. Appl. Microbiol.
Biotechnol. 90: 573-581.
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