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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

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

An Alkaline Family VIII Esterase from a Compost Metagenomic Library 317


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

318 Lee et al.


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.



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.

320 Lee et al.


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.



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.

322 Lee et al.


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

e Family V

III Esterase from

a Co

mp

ost M

etagen

om

ic Library

323

A 2016

⎪Vo

l. 26⎪

No. 0

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.


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.

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