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July 2014⎪Vol. 24⎪No. 7
J. Microbiol. Biotechnol. (2014), 24(7), 925–935http://dx.doi.org/10.4014/jmb.1402.02033 Research Article jmbReview
A Cold-Adapted Carbohydrate Esterase from the Oil-DegradingMarine Bacterium Microbulbifer thermotolerans DAU221: GeneCloning, Purification, and CharacterizationYong-Suk Lee1†, Jae Bok Heo2†, Je-Hoon Lee1, and Yong-Lark Choi1*
1Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea2Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Republic of Korea
Introduction
Carbohydrate esterases (CE) catalyze the O- or N-
deacylation of substituted saccharides. The vast diversity
of these enzymes, in terms of their substrate specificity and
structure, is reflected by the 16 different CE families in the
Carbohydrate-Active enZYmes (CAZy) [12]. A new family,
probably the seventeenth (CE17), will be added to include
the recently characterized Geobacillus stearothermophilus
acetyl esterase, which does not show any homology with
the established CE families [1]. Many of the enzymes that
do not fit into the established CE families have been
classified separately on the basis of their sequence similarities
[18]. Members of the CE6 family, which appears to include
enzymes with broad substrate specificity, are typical
serine-type esterases. Some members of the CE6 family
exhibit activities of other esterases, such as feruloyl
esterase [15], rhamnogalacturonan acetyl esterase, and
thioesterase [8, 28]. CE6-producing microbes usually
belong to the genera Neocallimastix [15], Orpinomyces [9],
and Fibrobacter [21, 46].
Marine environments contain over 100 different microbial
phyla, encompassing up to a billion different kinds of
marine microorganisms [10]. Microbulbifer thermotolerans is
a gram-negative, facultatively anaerobic, chemo-organotrophic
bacterium that belongs to the class gamma-proteobacteria.
It was isolated from Suruga Bay sediment samples in
Japan. M. thermotolerans colonies on marine agar are slightly
Received: February 18, 2014
Revised: March 24, 2014
Accepted: March 27, 2014
First published online
April 1, 2014
*Corresponding author
Phone: +82-51-200-7585;
Fax: +82-51-200-6536;
E-mail: [email protected]
†These authors contributed
equally to this work.
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2014 by
The Korean Society for Microbiology
and Biotechnology
A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6,
was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids
with the first 22 serving as a secretion signal peptide. The calculated molecular mass and
isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic
triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG,
QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST
belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight
α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the
characterization showed its optimal temperature and pH were 15°C and 8.0, respectively.
Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or
60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1 h. The results
suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was
increased by Na+ and Mg2+ ions but was strongly inhibited by Cu+ and Hg2+ ions, at all ion
concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM
and a kcat of 1.9 s-1. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192,
and His281) and Asp278 were essential for the enzyme activity.
Keywords: Cold-adapted enzyme, carbohydrate esterase, Microbulbifer thermotolerans,
purification, esterase, marine bacterium
926 Lee et al.
J. Microbiol. Biotechnol.
irregular, smooth, and brown. Although the bacterium can
grow at a NaCl concentration of 7%, optimal growth occurs
at concentrations of approximately 1-2%, and no growth
occurs in the absence of NaCl [30]. To date, only two kinds
of β-agarases have been reported from M. thermotolerans
[32, 33]. Hence, this is the first report on the production of a
carbohydrate esterase from M. thermotolerans.
The cold-adapted enzymes have the following three
advantageous properties that make them ideal for use in
biotechnological applications: (i) high activity, which
ensures optimum reactivity even at low concentrations of
the catalyst, thereby reducing the cost of the enzyme
preparation; (ii) cold activity, which preserves efficiency at
ambient temperatures, thereby avoiding the heating
process at both domestic and industrial levels; and (iii) heat
stability, which enables efficient and sometimes selective
inactivation by moderate heat input [29]. The yeast Candida
antarctica produces two cold-adapted lipases, A and B, the
latter being sold as Novozym435 by Novozymes (Denmark).
These enzymes are used in several organosynthesis
applications in food/feed processing and the production of
pharmaceuticals and cosmetics [6]. The xylanase from the
Antarctic bacterium Pseudoaltermonas haloplanktis is also a
key ingredient in industrial dough conditioners used to
improve bread quality [14]. In this study, the gene encoding
a putative cold-adapted family 6 carbohydrate esterase
(CEST) from the DAU221 strain of M. thermotoleran was
cloned, purified, and characterized. CEST exhibited cold-
adapted enzyme activity in the range of 5-20°C and
thermostability in the range of 50-70°C. To our knowledge,
CEST is the first M. thermotolerans cold-adapted carbohydrate
esterase reported as a potential biocatalyst for acyl-
degradation, carbohydrate bioconversion, and insecticide
degradation.
Materials and Methods
Bacterial Strains and Plasmids
Marine sediment samples were collected from an eastern coast(35°29.70’N, 129°26.11’E) in Korea. The samples were suspendedin marine broth 2216 (MA) (Difco, Detroit, MI, USA). Thesuspensions were suitably diluted with the broth and spread onmarine agar (Difco) containing 1% tributyrin emulsion (10 mMCaCl2, 20 mM NaCl, and 5% gum arabic solution) [23, 34],followed by incubation at 37°C for several days. One of thebacteria exhibiting tributyrin-degrading activity was chosen andnamed strain DAU221. Escherichia coli (E. coli) JM109 and EPI300-T1 were used as the cloning host, and BL21 (DE3) was used as theprotein expression host and grown at 37°C in Luria-Bertani (LB)
broth supplemented with ampicillin (50 µg/ml) or chloramphenicol(12.5 µg/ml) when required. Plasmids pUC118 and pCC1FOS(Epicentre, Madison, WI, USA) were used to construct thegenomic library, and pCold I (TaKaRa, Kyoto, Japan) was used asthe protein expression vector.
Phylogenetic Analysis by 16S rDNA
The polymerase chain reaction (PCR) was performed to amplifythe 16S rDNA coding region, using two oligonucleotide primers,5’-GAGTTTGATCCTGGCTCAG-3’ (positions 9 to 27 bp relativeto E. coli 16S rDNA) and 5’-AGAAAGGAGGTGATCCAGCC-3’(positions 1,525 to 1,542 bp relative to E. coli 16S rDNA) [43]. PCRwas performed using a TaKaRa PCR Thermal Cycler (Japan)programmed as follows: predenaturation for 60 sec at 95°C,30 cycles of denaturation at 95°C for 60 sec, annealing at 60°C at60 sec, and extension at 72°C for 90 sec, with a final extension at72°C for 10 min. The amplified 1.5 kb PCR products were clonedinto the pGEM T-easy vector (Promega, USA). Phylogenetic treeswere inferred using the ClustalX program [40].
Genomic Library Construction
A genomic library was constructed using a commercial fosmidlibrary construction kit, CopyControl Fosmid Library ProductionKit (Epicentre). A single colony of DAU221 was inoculated into10 ml of MB medium, incubated at 37°C, overnight on a rotaryshaker (180 rpm). Cells were harvested and genomic DNA wasprepared by the standard method, as described by Sambrook et al.[38]. The extracted DNA (1 µg) was efficiently sheared byhundreds of pipettings. It was treated for end-repair to generateblunt-end and 5’-phosphorylated DNA. End-repaired DNA waselectrophoresed in a 1% low-melting point agarose at 50 V for12 h, and DNA fragments over 40 kb were isolated from the gelusing GELase (Epicentre). The prepared DNA was ligated into thefosmid vector, pCC1FOS, and then the ligaton mixture waspackaged into lambda phages using MaxPlax Lambda PackagingExtracts (Epicentre). The packaged library was transducted intoE. coli EPI300-T1.
Screening of the Carbohydrate Esterase Gene
The genomic library was cultivated on LB agar plates withtributyrin and chloramphenicol. Any colonies with a clear zonearound each other were selected as acyl-degrading enzyme-producing recombinants, TB1~TB9. The plasmid DNA of theselected recombinant, TB3, was partially digested with XbaI andligated into pUC118 treated with XbaI enzyme and calf intestinalalkaline phosphatease (C.I.A.P). E. coli JM109 was transformedwith the ligation mixture by the Hanahan method [19]. The firstsubclones of the selected genomic library recombinant TB3 wereincubated on LB agar plates with tributyrin and ampicillin for 5days at 37°C. A positive clone, TB3Xb1, showed a clear zonearound colonies. The plasmid DNA of the first subclone, TB3Xb1,was partially digested with HindIII and ligated into the pUC118/
Cold-Adapted Carbohydrate Esterase from Microbulbifer thermotolerans DAU221 927
July 2014⎪Vol. 24⎪No. 7
HindIII/C.I.A.P. E. coli JM109 was transformed with the ligationmixture by the Hanahan method. The second subclones of theTB3Xb1 were incubated on LB agar plates with tributyrin andampicillin for 5 days at 37°C. A positive clone, TB3Xb1H2, showeda clear zone around the colony, and was selected as the acyl-degrading enzyme-producing clone and sequenced. Analysis ofsequenced data and sequenced similarity searches were performedusing the BLAST program of the National Center for BiotechnologyInformation (NCBI). Homology alignment was performed withthe CLUSTALW program [41] using MacVector 6.5 software(Oxford Molecular Group). The three-dimensional structure ofCEST was predicted using the PHYRE2 server (http://www.sbg.bio.ic.ac.uk/phyre2) [22].
Expression and Purification of CEST
The carbohydrate esterase from M. thermotolerans was expressedin a heterologous system in E. coli. The gene encoding a putativecarbohydrate esterase (CEST) was amplified with PCR using twoprimers that define the N-terminal without a signal peptide andC-terminal regions of the gene. The forward primer, TB3-CEST-SP, was used in the amplification with an EcoRI site (italics andunderline in the sequence): 5’-AGCACAGGAGAATTCGCTACCGAAGGCAAT-3’. The reverse primer, TB3-CEST-R, was usedwith a SalI site (italics and underline in the sequence): 5’-AGCGCACATATCGTCGACTTATTTACCGCA-3’. The reactionwas performed in a TaKaRa PCR thermal cycler (TaKaRa, Japan).The PCR product was double-digested by EcoRI and SalI andcloned into the expression vector pCold I with the same digestion.The recombinant was transformed into E. coli BL21 (DE3) (Novagen,Germany) for the protein expression. When the optical density at600 nm reached 0.4-0.5, 0.2 mM isopropyl-β-D-thiogalactoside(IPTG) was added, followed by incubation for 24 h at 15°C. Thecells were harvested by centrifugation at 6,000 rpm for 15 min at4°C, and then suspended with binding buffer (20 mM sodiumphosphate (pH 8.0), 0.5 M NaCl, and 5 mM imidazole). The cellswere disrupted by sonication (pulse-on 30 sec, pulse-off 30 sec,5 times, on ice), and the supernatant was collected by centrifugationat 13,000 rpm for 30 min at 4°C. The clear supernatant was loadedon to a HisTrap HP column (Amersharm Bioscience) equilibratedwith binding buffer and eluted with elution buffer (20 mMsodium phosphate (pH 8.0), 0.5 M NaCl, and 0.5 M imidazole) atthe flow rate of 1 ml/min. The eluted fractions were dialyzedovernight against 20 mM sodium phosphate (pH 8.0) andconcentrated by using Amicon Ultra-4 (Millipore, Bedford, MA,USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was carried out by the method of Laemmli [25]. Theconcentrated proteins were used for determining the enzymecharacterizations.
Enzyme Assay
The purified CEST activity was measured spectrophotometricallyusing p-nitrophenyl acetate (pNPA) (Sigma, St. Louis, MO, USA)
in a reaction mixture containing 20 mM sodium phosphate buffer(pH 8.0) and the purified enzyme in a final volume of 1 ml at 15°Cfor 15 min according to Winkler and Stuckman [45] with somemodifications [16]. The change in absorbance was measured overtime at 410 nm using an Ultrospec 2000 pro UV/visiblespectrophotometer (Amersham Bioscience).
The effect of pH on the activity of CEST was measured over apH range of 2.5-10.6. The buffers used were 20 mM citrate buffer(pH 3.0-5.6), 20 mM sodium phosphate buffer (pH 6.0-8.0),20 mM Tris-HCl buffer (pH 7.5-9.0), and 20 mM glycine-NaOHbuffer (pH 8.6-10.6). The optimal temperature of the CEST wasdetermined by measuring the enzyme activity at various temperatures(5-70°C) in 20 mM sodium phosphate buffer (pH 8.0). For thetemperature stability assay, the enzyme was preincubated withvarious buffers at 4°C without substrate for 30 min. The extremethermostability was determined by preincubating the CEST in20 mM sodium phosphate buffer (pH 8.0) at various temperatures(50-70°C) for various hours (1-6 h). The effects of potentialinhibitors or activators on the enzyme were determined by theaddition of various metal salts to the reaction mixture at a finalconcentration of 1, 5, or 10 mM, which was preincubated for10 min at 4°C. The test ions were BaCl2, CaCl2, CsCl, CuCl2, FeCl2,HgCl2, KCl, LiCl, NaCl, NiCl2, MgCl2, MnCl2, ZnCl2, and EDTA.The remaining activity was assayed as described above. Fordetermination of the Km and kcat values, the assays containedsubstrates at concentrations of 0.1-1 mM. Kinetic parameterswere obtained from the Lineweaver-Burk plots against varioussubstrate concentrations using SWIFT II Applications software(Amersham Bioscience).
Site-Directed Mutagenesis
Site-directed mutagenesis was carried out using a QuikChangeXL site-directed mutagenesis kit (Stratagene), according to themanufacturer’s instructions. Primers used in the site-directedmutagenesis study are presented in Table 1.
Nucleotide Sequence Accession Numbers
The nucleotide sequences of the 16S rDNA and carbohydrateesterase gene reported in this article were assigned as GenBankaccession numbers KC571186 and KC571187.
Table 1. Primers used for site-directed mutagenesis.
Primer Sequence
S37G 5’-AGGCGGCCAGGGGAACATGGAGGGGTATGGG-3’
M189H 5’-GGGATCGTATGGCACCAAGGTGAGGCCGAT-3’
E192N 5’-CGTATGGATGCAAGGTAACGCCGATGCCTTTG-3’
E192A 5’-CGTATGGATGCAAGGTGCGGCCGATGCCTTTG-3’
D278N 5’-GGTTACCTAGACAACGGTTGGCACTACAATACCG-3’
D278A 5’-GGTTACCTAGACGCGGGTTGGCACTACAATACCG-3’
H281Q 5’-GACGACGGTTGGCAGTACAATACCGAAGGC-3’
928 Lee et al.
J. Microbiol. Biotechnol.
Results and Discussion
Isolation and Identification of the Strain DAU221
Approximately 100 different bacterial strains were isolated
from the marine sediment in Korea. Strain DAU221 was
detected in a clear zone around the colonies on the MB-
tributylin plate. Therefore, strain DAU221 was isolated as a
candidate acyl-degrading enzyme-producing bacterium.
This strain is a Gram-negative rod-shaped bacterium that
produces a brown pigment at 5 days after incubation at
37°C. Agar, xylan, colloidal chitin, casein, soluble starch,
and esculin were hydrolyzed by strain DAU221 (data not
shown). The phylogenetic position was determined by
comparing the 16S rDNA sequence. Sequence similarities
were of 99% for M. thermotolerans JAMB A94 (11); 97% for
M. chitinilyticus ABABA212 and M. maritimus TF-17; 96%
for M. donhaiensis CN85, M. epialgicus F-104, M. halophilus
YIM91118, M. okinawensis ABABA211 and ABABA23, and
M. variabilis Ni-2088; 95% for M. agarilyticus JAMB A3,
M. celer ISL-39, and M. salipaludis SM-1; and 94% for
M. hydrolyticus DSM11525 and M. elongates ATCC 10144.
The phylogenetic tree, based on the comparison of the 16S
rDNA sequences, is shown in Fig. 1. Based on these data,
the strain DAU221 was identified as M. thermotolerans.
Identification of a Novel Carbohydrate Esterase from M.
thermotolerans
Twenty thousand fosmid clones were obtained using a
Fosmid Library Production Kit. Many transformants
showed hydrolytic activity when cultured on LB-tributylin-
chloramphenicol plates. Some of these transformants were
named as TB1-TB9. A selected clone, TB3, was partially and
sequentially digested with XbaI and HindIII to obtain full
nucleotide sequences of the tributylin-hydrolyzing enzyme
from M. thermotolerans DAU221. The TB3 fragment, which
was obtained after digestion with XbaI and HindIII, was
composed of 4,473 nucleotides and showed six open reading
frames (ORFs), which collectively encoded for more than
100 amino acids. The coding regions within ORF1 were
found to encode iduronate-2-sulfatase of Planctomyces maris
DSM 8797 (GenBank Accession No. ZP_01856700.1), CE of
Flavobacteriaceae bacterium S85 (GenBank Accession No.
Fig. 1. Phylogenetic tree based on 16S rDNA sequences,
showing the positions of DAU221 in relation to strains of
recognized Microbulbifer species.
Bacillus atrophaeus 16S rDNA (AB021181) was used as an outgroup.
Bar, 0.1 substitution per nucleotide position.
Table 2. BLASTP results of each ORF from the TB3/XbaI/HindIII fragment in GenBank.
ORFs Start Stop bp aa BLASTP results Identity (%) Accession Number
ORF1 1298 2221 924 308 Carbohydrate esterase 32 ZP_09498014.1
Iduronate-2-sulfatase 49 ZP_01856700.1
ORF2 4415 3687 729 243 Hypothetical protein 68 YP_007273880.1
ORF3 834 136 699 233 Hypothetical protein 36 ZP_09754815.1
ORF4 3132 2470 663 221 Hypothetical protein 57 ZP_10134161.1
SNF2-related protein 25 ZP_02179755.1
ORF5 3534 3154 381 127 Hypothetical protein 49 YP_432747.1
Orotidine 5’-phosphate decarboxylase 33 YP_004661824.1
ORF6 1729 1394 336 112 ABC-type transporter 31 ZP_15537327.1
bp, base pairs; aa, amino acids.
Cold-Adapted Carbohydrate Esterase from Microbulbifer thermotolerans DAU221 929
July 2014⎪Vol. 24⎪No. 7
ZP_09498014.1), and CE of Zobellia galactanivorans
(GenBank Accession No. YP_004737923.1); the amino acid
identities with these enzymes were 49%, 32%, and 31%,
respectively (Table 2).
The CEST gene, cest, in ORF1 begins with an ATG at
nucleotide 1298 and ends with a TAA at nucleotide 2221
(Fig. 2). A putative ribosome-binding site of 5’-AGGAG-3’
presents 7 bp upstream from the initiation codon ATG. The
5’-TTGGCC-3’ for the -35 region and the 5’-ATTAAT-3’ for
Fig. 2. Nucleotide sequences and deduced amino acid
sequences of the carbohydrate esterase gene from M.
thermotolerans DAU221.
The possible -35 and -10 sequences in the promoter region are
indicated and the possible ribosome-binding site is the quadrangle. The
signal peptide predicted from the SignalP site is indicated with an
underline. Symbols above the sequences represent the second structure,
pink quadrangles represent α-helices, and blue arrows represent the
β-strand.
Fig. 3. Phylogenetic tree of CEST from M. thermotolerans
DAU221 and other carbohydrate esterases.
The tree was constructed by the use of ClustalX software. The scale
bar represents 0.1 substitution per amino acid position.
930 Lee et al.
J. Microbiol. Biotechnol.
the -10 region were located 80 bp upstream from the
initiation codon with 19 bp spacing. Thus, the CE gene
from DAU221 is 864 bp, and encodes a protein with 307
amino acids. The signal peptide sequence was analyzed
using the SignalP server (http://www.cbs.dtu.dk/services/
SignalP). The most likely cleavage sites are presumed to be
between Ala-21 and Ala-22 [35]. Hence, the mature protein
was predicted to contain 286 amino acids with an
estimated molecular mass of 31,244 Da and an isoelectric
point of 4.73.
The deduced amino acid sequence of CEST was
compared with other CE6 sequences using the BLASTP
program [3]. Using the sequences for CE families 1-16,
obtained from the CAZy Database, a phylogenetic tree was
constructed (Fig. 3). The enzyme showed identities and
similarities with other bacterial CE6, such as those of
Roseobacter denitrificans (ABI93412.1, 25% identity, 35%
similarity), Fibrobacter succinogenes ABL25018 (27%, 42%)
and ADL27361 (21%, 36%), Prevotella ruminicola (ADE82678,
23%, 38%), Zunongwangia profunda (ADF51369, 22%, 39%),
Paludibacter propionicigenes (ADQ79784, 23%, 35%), Cytophaga
hutchinsonii (ABG58511, 21%, 39%), Neocallimastix patriciarum
(AAB69090, 23%, 38%), Orpinomyces sp. (AAC14690, 23%,
38%), Spirosoma linguale (ADB38573, 25%, 38%), Leadbetterella
byssophila (ADQ16128, 23%, 39%), Chitinophaga pinensis
(ACU64246, 26%, 41%), Alkaliphilus metalliredigens (ABR50009,
21%, 36%), and Bacillus amyloliquefaciens (ABS74765, 25%,
39%).
Fig. 4 shows a representative portion of the multiple
alignments. The three stretches of the conserved residues are
GQSNMXG, QGEX(D/N), and DXXH. The enzymes of the
CE6 family possess a catalytic triad consisting of the serine,
glutamate, and histidine residues [7, 8, 28]. The catalytic
triad of CEST was identified in the structure containing the
residues Ser37, Glu192, and His281. Ser37 and Gln38 are
known to be involved in the formation of the oxyanion
hole. Gln190, Gly191, and Glu192 are known to be crucial
for the proper positioning of Gln38 through a hydrogen-
bond network [8]. The three-dimensional structure of CEST
was predicted using the PHYRE2 server [22]. CEST belongs
to the SGNH hydrolase superfamily, with a 6-stranded β-
sheet and an 8-stranded α-helix (Fig. 5). The enzymes of the
SGNH hydrolase superfamily facilitate the hydrolysis of
the ester, thioester, and amide bonds in a range of substrates
including complex polysaccharides, lysophospholipids,
and acyl-CoA esters [8]. PHYRE search for homologous
Fig. 4. Alignment of the deduced amino acid to the carbohydrate esterase from various microorganisms.
The deduced amino acid of the carbohydrate esterase genes from M. thermotolernas DAU221 (CEST) was compared with ABI93412, R. denitrificans;
ABL25018 and ADL27361, F. succinogenes; ADE82678, P. ruminicola; ADF51369, Z. profunda; ADQ79784, Pa. propionicigenes; ABG58511, C.
hutchinsonii; AAB69090, N. patriciarum; AAC14690, Orpinomyces sp.; ADB38573, S. linguale; ADQ16128, L. byssophila; ACU64246, Ch. pinensis;
ABR50009, A.metalliredigens; and ABS74765, B. amyloliquefaciens.
Fig. 5. Ribbon diagram showing the secondary structure of
CEST with rainbow coloring from the amino-terminus (blue)
to the carboxyl-terminus (red).
Cold-Adapted Carbohydrate Esterase from Microbulbifer thermotolerans DAU221 931
July 2014⎪Vol. 24⎪No. 7
structures in PDB identified the SGNH hydrolase structure;
Clostridium acetobutylicum (PDB code 1ZMB, 23% identity),
Arabidopsis thaliana (PDB code 2APJ, 23% identity), and
E. coli O157:H7 (PDB code 3PT5, 20% identity).
Expression and Purification of Recombinant CEST
The recombinant CEST was overexpressed in E. coli BL21
(DE3) using pCold I as the expression vector and purified
by His-tag affinity chromatography. The purified enzyme
gave a single band on SDS-PAGE. The molecular mass of
the denatured enzyme was approximately 31 kDa, which
was in agreement with the molecular mass deduced from
the amino acid sequence (31,244 Da) (Fig. 6).
Properties of Recombinant CEST
The optimum activity of CEST was measured over a pH
range of 2.5-10.6 and a temperature range of 4-70°C, with
p-nitrophenyl acetate as the substrate. The optimum pH
was found to be 8.0 (Fig. 7A). With sodium phosphate
buffer (2.92 µmol/mg/min) it was 1.17 times that obtained
with Tris-HCl buffer (2.49 µmol/mg/min). CEST exhibited
activity at low temperatures (4-15°C), with the maximum
activity observed at 15°C. On ice, this enzyme maintained
89% of its maximal activity (at 15°C) (Fig. 7B). Some of the
cold-adapted carbohydrate esterases were described
Fig. 6. SDS-PAGE analysis of CEST.
Lane 1: molecular weight marker. Lane 2: cell-free extract. Lane 3:
purified CEST.
Fig. 7. Effects of pH and temperature on the activity of CEST.
(A) Optimum pH of CEST. pH range; 20 mM citrate buffer (pH 3.0-
5.6), 20 mM sodium phosphate buffer (pH 6.0-8.0), 20 mM Tris-HCl
buffer (pH 7.5-9.0), and 20 mM glycine-NaOH buffer (pH 8.6-10.6).
(B) Optimum temperature (black circle) and temperature stability (white
circle) of CEST. Enzyme was preincubated at each temperature for
30 min for checking the enzyme stability. (C) Extreme-temperature
stability of CEST. Enzyme was preincubated for various times (1, 3, or 6 h)
at 50oC (black triangle), 60oC (black quardangle), or 70°C (black circle).
932 Lee et al.
J. Microbiol. Biotechnol.
previously. These were pH 8.0 and 20°C for cold-adapted
PSHAa from Pseudoalteromonas halohplanktis TAC125 [4],
7.5 and 25°C for EstO enzyme from Pseudoalteromonas
arctica [2], 7.5 and 25°C for AELH from Acinetobacter sp.
strain no. 6 [39], and 8.0 and 15-20°C for OLEI01171 from
Oleispira antarctica [26]. These cold-adapted CEs showed
30-90% decrease in their activity at 5°C. However, CEST
showed 1.5% decrease in its maximal activity at 5°C and
11% decrease on ice. The activity of CEST was slightly
reduced at 20°C, and it became insignificant at 60°C.
CEST exhibited thermostability within a broad range of
temperatures from 10°C to 40°C. The preincubated CEST at
25°C for 30 min showed its maximal activity (3.20 µmol/
mg/min) and maintained 40-50% activity at 50-60°C for
30 min. Furthermore, CEST was preincubated for 1, 3, or
6 h at 50°C, 60°C, or 70°C for each incubation time (Fig.
7C). These preincubated CESTs maintained 95%, 91%, or
89% activity for 1 h; 77%, 81%, or 41% for 3 h; and 74%,
70%, or 16% activity for 6 h at 50°C, 60°C, or 70°C. At 50°C
and 60°C, the results indicated that the preincubation of
CEST for 6 h helped maintain 70% or more of its maximal
activity. At 70°C, the results indicated that the activity
gradually decreased. Ohgiya et al. [31] have described three
groups of cold-adapted enzymes according to their
thermolability and catalytic properties. Group 1 has similar
activity and more heat sensitivity than the equivalent
mesophilic enzymes. Group 2 has higher activity at low
temperatures and more heat sensitivity. Group 3 has higher
activity at low temperatures and similar thermostability
[26]. Therefore, CEST probably belongs to group 3 of the
cold-adapted enzymes, because it shows similar activity
and thermostability.
To clarify the effect of various metal ions and reagents on
CEST activity, the enzyme assays were carried out in the
presence of Ba2+, Ca2+, Co2+, Cs2+, Cu2+, Fe3+, Hg2+, K+, Li2+,
Mg2+, Mn2+, Na+, Ni2+, Zn2+, or EDTA at final concentrations
of 1, 5, or 10 mM (Table 3). When Na+, Mg2+, and EDTA
reagent were added, the enzyme activity increased to
125%, 117%, or 109% at ion concentrations of 1, 5, or
10 mM, respectively. The metal ions Co+ and Zn2+ increased
the enzyme activity at low ion concentrations, but strongly
decreased the enzyme activity at high ion concentrations.
Moreover, the enzyme activity was inhibited by Ba2+, Ca2+,
Cs2+, Fe3+, K+, Li+, Mn2+, and Ni2+ at 1, 5, or 10 mM,
respectively. In particular, Cu2+ and Hg2+ ions strongly
inhibited the enzyme activity at all concentrations. According
to the most recent reports, the levels of a PE10 from the
marine bacterium Pelagibacterium halotolerans B2T increased
in the presence of NaCl and showed maximal activity at
3 M NaCl [20]. A PDF1Est from Anoxybacillus sp. PDF1 was
strongly inhibited by the presence of Co+ and Zn2+, whereas
the presence of Ca2+ led to mild activation [5]. A PsyEst
from Psychrobacter sp. Ant300 was inhibited by Mg2+ and
Mn2+, whereas its activity was enhanced in the presence of
Ca2+ [24]. The activity of PSHAa1385 from P. haloplanktis
TAC125 has been reported to increase in the presence of
Ca2+, Mg2+, and Mn2+ [4]. The activity of Axe6A from
Fibrobacter succinogenes decreased by more than 50% in the
presence of Fe2+, Cu2+, and Zn2+ at a concentration of 1 mM,
but was unaffected by Mn2+, Co2+, Ca2+, Mg2+, and EDTA at
a concentration of 10 mM. In contrast, the activity of Axe6B
was inhibited by all ions, except Ca2+, at 10 mM [21]. To
understand the basic catalytic parameters of CEST, steady-
state kinetic analysis was performed. CEST had Km and kcat
values of 0.278 mM and 1.9 s-1, respectively, when pNPA
was used as the substrate.
Mutational Studies of CEST
CEST has three residues (Ser37, Glu192, and His281) that
are highly conserved among the CE6 family proteins.
Glutamate increases the pKa of its imidazole nitrogen in
histidine. This allows the histidine to become a strong
general base by removing a proton from the hydroxyl
group of serine. The deprotonated serine acts as a
nucleophile and attacks the carbonyl carbon of the acetyl
group [28, 46]. In order to probe the role of the three
Table 3. Effects of various metal ions on the activity of CEST.
1 mM 5 mM 10 mM
Na+ 126 ± 1 125 ± 1 123 ± 2
Mg2+ 117 ± 1 117 ± 1 117 ± 2
EDTA 109 109 ± 1 109 ± 1
Co2+ 124 ± 3 125 ± 2 14 ± 7
Zn2+ 111 ± 1 53 ± 5 N.D.
Ca2+ 89 ± 1 89 ± 2 86 ± 3
Cs2+ 72 ± 1 70 ± 3 70
Ba2+ 67 ± 2 72 68
Li2+ 60 ± 1 65 ± 1 63 ± 2
K+ 59 ± 4 62 ± 1 62
Ni2+ 87 ± 2 78 ± 3 ND
Mn2+ 76 71 ND
Fe3+ 47 ± 5 ND ND
Cu2+ ND ND ND
Hg2+ ND ND ND
Data are shown as means ± standard errors.
ND, not detected.
Cold-Adapted Carbohydrate Esterase from Microbulbifer thermotolerans DAU221 933
July 2014⎪Vol. 24⎪No. 7
residues in the active site of CEST, we mutated these
residues to alanine or other residues. These conserved
residues were replaced by S37G, E192N, E192A, and
H281Q. These mutations led to a complete loss of enzyme
activity, which was in agreement with the findings of
previous studies [15, 28, 44]. These results support the view
that these residues are essential for the enzyme activity.
Yosida et al. [46] suggested that the aspartate residue of
FSUAxe6B from F. succinogenes S85 contributes to the
catalysis as helper acids, similar to the glutamate residue in
the conserved region. The Asp278 residue of CEST was
replaced by Gln and Ala. The D278N and D278A mutants
of CEST lost much of their enzyme activity. Bitto et al. [8]
reported that the two conserved residues in CE6 family
proteins are GQSNMXG and QGEX(D/N). Similarly,
Lopez-Cortes et al. [28] mentioned that the three characteristic
motifs of the CE6 family are G(D/Q)SX, HQGE, and
DXXH. The His residue in the HQGE motif reported by
Lopez-Cortes et al. was replaced by Met189 in CEST.
M189H was constructed to clarify the role of the His in the
HQGE motif. When M189H was compared with pure
CEST, the enzyme activities were found to be similar (Fig.
8). This result suggests that the His residue in the HQGE
motif is highly conserved, but not essential for the enzyme
activity.
Application of CEST
CEs are important in the hydrolysis of numerous
endogenous and xenobiotic ester-containing compounds,
such as carbamates, organophosphorus pesticides, and
pyrethroids [42]. Pyrethroids have been used for more than
30 years and are the most commonly used insecticides in
the world. However, use of these pesticides has caused
many problems, such as pest resistance, soil and water
contamination, and health hazards arising from human
exposure to pyrethroids. Microbial degradation plays an
important role in the elimination of these pesticides [47].
Recently, several CEs were identified from Aspergillus niger
ZD11 [27], Sphingobium sp. JZ-1 [42], and Ochrobactrum
anthropic YZ-1 [47], etc.
The demand for active biocatalysts that could be used
under extreme conditions (low or high temperatures, acidic
or basic solutions, or high salt contents) has increased in
many biotechnology industries. Therefore, the isolation of
biotechnologically relevant enzymes from extremophilic
microbes has become a challenging task in recent years [2,
11, 36, 37]. Cold-adapted enzymes offer economic benefits
through energy savings, because they negate the requirement
for expensive heating steps. In addition, these enzymes
function in cold environments, as well as during the winter
season, and provide increased reaction yields and high
stereospecificity, while minimizing undesirable chemical
reactions that can occur at higher temperatures. Moreover,
the thermal lability of these enzymes allows rapid and
simple enzyme inactivation [13, 17]. CEST, a cold-adapted
CE, from M. thermotolerans DAU221, could be useful in
removing pyrethroid residues from soil, sediment, and
agricultural products for bioremediation.
Acknowledgments
This research was supported by the Basic Science Research
Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science
and Technology (2011-0008619) and “Cooperative Research
Program for Agriculture Science & Technology Development
(Projects No.PJ009759)”, Rural Development Administration,
Republic of Korea to J.B.H.
References
1. Alalouf O, Balazs Y, Volkinshtein M, Grimpel Y, Shoham G,Shoham Y. 2011. A new family of carbohydrate esterases isrepresented by a GSDL hydrolase/acetylxylan esterase fromGeobacillus stearothermophilus. J. Biol. Chem. 86: 41993-42001.
2. Al Khudary R, Venkatachalam R, Katzer M, Elleuche S,Antranikian G. 2010. A cold-adapted esterase of a novelmarine isolate, Pseudoalteromonas arctica: gene cloning, enzymepurification and characterization. Extremophiles 14: 273-285.
3. Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W,Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic
Acids Res. 25: 3389-3402.4. Aurilia V, Parracino A, Saviano M, Rossi M, D’Auria S.
2007. The psychrophilic bacterium Pseudoalteromonas halosplanktis
TAC125 possesses a gene coding for a cold-adapted feruloyl
Fig. 8. Activity analysis of the site-directed mutants of CEST.
934 Lee et al.
J. Microbiol. Biotechnol.
esterase activity that shares homology with esterase enzymesfrom γ-proteobacteria and yeast. Gene 397: 51-57.
5. Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. 2011.Cloning, purification and characterization of a thermostablecarboxylesterase from Anoxybacillus sp. PDF1. Protein Expr.
Purif. 80: 74-79.6. Babu J, Ramteke PW, Thomas G. 2008. Cold active microbial
lipases: some hot issues and recent developments. Biotechnol.
Adv. 26: 457-470.7. Biely P. 2012. Microbial carbohydrate esterases deacetylating
plant polysaccharides. Biotechnol. Adv. 30: 1575-1588.8. Bitto E, Bingman CA, McCoy JG, Allard STM, Wesenberg E,
Phillips Jr GN. 2005. The structure at 1.6 Å resolution of theprotein product of the At4g34215 gene from Arabidopsis
thaliana. Acta Crystallogr. D Biol. Crystallogr. D61: 1655-1661.9. Blum DL, Li XL, Chen H, Ljungdahl LG. 1999. Characterization
of an acetyl xylanesterase from the anaerobic fungusOrpinomyces sp. strain PC-2. Appl. Environ. Microbiol. 65:
3990-3995.10. Burgess JG. 2012. New and emerging analytical techniques
for marine biotechnology. Curr. Opin. Biotechnol. 23: 29-33.11. Burton SG, Cowan DA, Woodley JM. 2002. The search for
the ideal biocatalyst. Nat. Biotechnol. 20: 37-45.12. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard
V, Henrissat B. 2009. The Carbohydrate-Active Enzymesdatabase (CAZy): an expert resource for glycogenomics.Nucleic Acids Res. 37: D233-D238.
13. Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002.Low-temperature extremophiles and their applications. Curr.
Opin. Biotechnol. 13: 253-261. 14. Collins T, De Vos D, Hoyoux A, Savvides SN, Gerday C,
van Veeumen J, Feller G. 2005. Study of the active siteresidues of a glycoside hydrolase family 8 xylanase. J. Mol.
Biol. 354: 425-435.15. Darlymple BP, Cybinski DH, Layton I, McSweeney CS, Xue
GP, Swadling YJ, Lowry JB. 1997. Three Neocallimastix
partiarum esterases associated with the degradation ofcomplex polysaccharides are members of a new family ofhydrolases. Microbiology 143: 2605-2614.
16. Dheeman DS, Henehan GTM, Frias JM. 2011. Purificationand properties of Amycolatopsis mediterranei DSM 43304lipase and its potential in flavor ester synthesis. Bioresour.
Technol. 102: 3373-3379.17. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P,
Collins T, et al. 2000. Cold-adapted enzymes: from fundamentalsto biotechnology. Trends Biotechnol. 18: 103-107.
18. Gupta Udtha DBRK, Kouskoumvekaki I, Solsson L, PanagiotouG. 2011. The interplay of descriptor-based computationalanalysis with pharmacophore modeling builds the basis fora novel classification scheme for feruloyl esterases. Biotechnol.
Adv. 29: 94-110. 19. Hanahan D. 1983. Studies on transformation of Escherichia
coli with plasmids. J. Mol. Biol. 166: 557-580.
20. Jiang X, Huo Y, Cheng H, Zhang X, Zhu X, Wu M. 2012.Cloning, expression and characterization of a halotolerantesterase from a marine bacterium Pelagibacterium halotolerans
B2T. Extremophiles 16: 427-435.21. Kam DK, Jun HS, Ha JK, Forsberg CW. 2005. Characteristics
of adjacent family 6 acetylxylan esterases from Fibrobacter
succinogenes and the interaction with the Xyn10E xylanase inhydrolysis of acetylated xylan. Can. J. Microbiol. 51: 821-832.
22. Kelly LA, Sternberg MJE. 2009. Protein structure predictionof the web: a case study using the PHYRE server. Nat.
Protoc. 4: 363-371.23. Kim SH, Park IH, Lee SC, Lee YS, Zhou Y, Kim CM, et al.
2008. Discovery of three novel lipase (lipA1, lipA2, and lipA3)and lipase-specific chaperone (lipB) genes present inAcinetobacter sp. DYL129. Appl. Microbiol. Biotechnol. 77:
1041-1051.24. Kulakova L, Galkin A, Nakayama T, Esaki N. 2004. Cold-
active esterase from Psychrobacter sp. Ant300: gene cloning,characterization, and the effects of Gly → Pro substitutionnear the active site on its catalytic activity and stability.Biochim. Biophys. Acta 169: 59-65.
25. Laemmli UK. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 227:
680-685.26. Lemak S, Tchigvintsev A, Petit P, Flick R, Singer AU,
Brown G, et al. 2012. Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171from the oil-degrading marine bacterium Oleispira antarctica.Biochem. J. 445: 193-203.
27. Liang WQ, Wang ZY, Li H, Wu PC, Hu JM, Luo N, et al.2005. Purification and characterization of a novel pyrethroidhydrolase from Aspergillus niger ZD11. J. Agric. Food Chem.
53: 7415-7420. 28. Lopez-Cortes N, Reyes-Duarte D, Beloqui A, Polaina J,
Ghazi I, Golishina OV, et al. 2007. Catalytic role ofconserved HQGE motif in the CE6 carbohydrate esterasefamily. FEBS Lett. 581: 4657-4662.
29. Margesin R, Feller G. 2010. Biotechnological applications ofpsychrophiles. Environ. Technol. 31: 835-844.
30. Miyazaki M, Nogi Y, Ohta Y, Hatada Y, Fujiwara Y, Ito S,Horikoshi K. 2008. Microbulbifer agarlyticus sp. nov. andMicrobulbifer thermotolerans sp. nov., agar-degrading bacteriaisolated from deep-sea sediment. Int. J. Syst. Evol. Microbiol.
58: 1128-1133.31. Ohgiya S, Hoshino T, Okuyama H, Tanaka S, Ishizaki K. 1999.
Biotechnology of enzymes from cold-adapted microorganisms,pp. 17-34. In Margesin R, Schinner F (eds.). Biotechnological
Applications of Cold-Adapted Organisms, Heidelberg, Springer-Verlag.
32. Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004.Cloning, expression, and characterization of a glycosidehydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66: 266-275.
Cold-Adapted Carbohydrate Esterase from Microbulbifer thermotolerans DAU221 935
July 2014⎪Vol. 24⎪No. 7
33. Ohta Y, Nogi Y, Miyazaki M, Li Z, Hatada Y, Ito S,Horikoshi K. 2004. Enzymatic properties and nucleotide andamino acid sequences of a thermostable β-agarase from thenovel marine isolate, JAMB-A94. Biosci. Biotechnol. Biochem.
68: 1073-1081.34. Park IH, Kim SH, Lee YS, Lee SC, Zhou Y, Kim CM, et al.
2009. Gene cloning, purification, and characterization of acold-adapted lipase produced by Acinetobacter baumannii
BD5. J. Microbiol. Biotechnol. 19: 128-135.35. Peterson TN, Brunak S, von Heijne G, NielsenH. 2011.
SignalP 4.0: discriminating signal peptides from transmembraneregions. Nat. Methods 8: 785-786.
36. Rao L, Zhao X, Pan F, Li Y, Xue Y, Ma Y, Lu JR. 2009.Solution behavior and activity of a halophilic esterase underhigh salt concentration. PLoS One 4: e6980.
37. Salameh MA, Wiegel J. 2007. Purification and characterizationof two highly thermophilic alkaline lipases from Thermosyntropha
lipolytica. Appl. Environ. Microbiol. 73: 7725-7731.38. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning:
A Laboratory Manual, 2nd Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.
39. Suzuki T, Nakayama T, Kurihara T, Nishino T, Esaki N.2002. A cold-active esterase with a substrate preference forvinyl esters from a psychrotroph, Acinetobacter sp. strain no.6: gene cloning, purification, and characterization. J. Mol.
Catal. B Enzym. 16: 255-263.40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,
Higgins DG. 1997. The ClustalX Windows interface: flexiblestrategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acids Res. 24: 4876-4882.41. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequencealignment through sequence weighting, positions-specificgap penalties and weight matrix choice. Nucleic Acids Res.
22: 4673-4680.42. Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. 2009. Cloning
of a novel pyrethroid-hydrolyzing carboxylesterase genefrom Sphingobium sp. strain JZ-1 and characterization of thegene product. Appl. Environ. Microbiol. 75: 5496-5500.
43. Weisburg WG, Barns SM, Pelletire DA, Lane DJ. 1991. 16Sribosomal DNA amplication for phylogenetic study. J. Bacteriol.173: 697-703.
44. Wei Y, Schottel JL, Derewenda U, Swenson L, Patkar S,Derewenda S. 1995. A novel variant of the catalytic triad inthe Streptomyces scabies esterase. Nature 2: 218-223.
45. Winkler UK, Stuckman M. 1979. Glycogen, hyaluronate andsome other polysaccharides greatly enhance the formationof exolipase by Serratia marcescens. J. Bacteriol. 138: 663-679.
46. Yoshida S, Mackie RI, Cann IKO. 2010. Biochemical anddomain analyses of FSUAxe6B, a modular acetyl xylanesterase, identify a unique carbohydrate binding module inFibrobacter succinogenes S85. J. Bacteriol. 192: 483-493.
47. Zhai Y, Li K, Song JL, Shi YH, Yan YC. 2012. Molecularcloning, purification and biochemical characterization of anovel pyrethroid-hydrolyzing carboxylesterase gene fromOchrobactrum anthropi YZ-1. J. Hazard. Mater. 221-222: 206-212.
