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A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization
A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 925-935
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 18, 2014
  • Accepted : March 27, 2014
  • Published : July 28, 2014
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About the Authors
Yong-Suk Lee
Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea
Jae Bok Heo
Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Republic of Korea
Je-Hoon Lee
Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea
Yong-Lark Choi
Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea
ylchoi@dau.ac.kr

Abstract
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℃ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50℃ or 60℃ for 6 h, and 89% of its enzyme activity when preincubated at 70℃ for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na + and Mg 2+ ions but was strongly inhibited by Cu + and Hg 2+ ions, at all ion concentrations. Using p -nitrophenyl acetate as a substrate, the enzyme had a K m of 0.278 mM and a k cat 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
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 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 heatingprocess 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℃ and thermostability in the range of 50-70℃. To our knowledge, CEST is the first M. thermotolerans cold-adapted carbohydrate esterase reported as a potential biocatalyst for acyldegradation, 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 suspended in marine broth 2216 (MA) (Difco, Detroit, MI, USA). The suspensions were suitably diluted with the broth and spread on marine agar (Difco) containing 1% tributyrin emulsion (10 mM CaCl 2 , 20 mM NaCl, and 5% gum arabic solution) [23 , 34] , followed by incubation at 37℃ for several days. One of the bacteria exhibiting tributyrin-degrading activity was chosen and named strain DAU221. Escherichia coli ( E. coli ) JM109 and EPI300- T1 were used as the cloning host, and BL21 (DE3) was used as the protein expression host and grown at 37℃ 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 the genomic library, and pCold I (TaKaRa, Kyoto, Japan) was used as the protein expression vector.
- Phylogenetic Analysis by 16S rDNA
The polymerase chain reaction (PCR) was performed to amplify the 16S rDNA coding region, using two oligonucleotide primers, 5’-GAGTTTGATCCTGGCTCAG-3’ (positions 9 to 27 bp relative to E. coli 16S rDNA) and 5’-AGAAAGGAGGTGATCCAGCC-3’ (positions 1,525 to 1,542 bp relative to E. coli 16S rDNA) [43] . PCR was performed using a TaKaRa PCR Thermal Cycler (Japan) programmed as follows: predenaturation for 60 sec at 95℃,30 cycles of denaturation at 95℃ for 60 sec, annealing at 60℃ at 60 sec, and extension at 72℃ for 90 sec, with a final extension at 72℃ for 10 min. The amplified 1.5 kb PCR products were cloned into the pGEM T-easy vector (Promega, USA). Phylogenetic trees were inferred using the ClustalX program [40] .
- Genomic Library Construction
A genomic library was constructed using a commercial fosmid library construction kit, CopyControl Fosmid Library Production Kit (Epicentre). A single colony of DAU221 was inoculated into 10 ml of MB medium, incubated at 37℃, overnight on a rotary shaker (180 rpm). Cells were harvested and genomic DNA was prepared by the standard method, as described by Sambrook et al . [38] . The extracted DNA (1 μg) was efficiently sheared by hundreds of pipettings. It was treated for end-repair to generate blunt-end and 5’-phosphorylated DNA. End-repaired DNA was electrophoresed in a 1% low-melting point agarose at 50 V for 12 h, and DNA fragments over 40 kb were isolated from the gel using GELase (Epicentre). The prepared DNA was ligated into the fosmid vector, pCC1FOS, and then the ligaton mixture was packaged into lambda phages using MaxPlax Lambda Packaging Extracts (Epicentre). The packaged library was transducted into E. coli EPI300-T1.
- Screening of the Carbohydrate Esterase Gene
The genomic library was cultivated on LB agar plates with tributyrin and chloramphenicol. Any colonies with a clear zone around each other were selected as acyl-degrading enzyme-producing recombinants, TB1~TB9. The plasmid DNA of the selected recombinant, TB3, was partially digested with XbaI and ligated into pUC118 treated with XbaI enzyme and calf intestinal alkaline phosphatease (C.I.A.P). E. coli JM109 was transformed with the ligation mixture by the Hanahan method [19] . The first subclones of the selected genomic library recombinant TB3 were incubated on LB agar plates with tributyrin and ampicillin for 5 days at 37℃. A positive clone, TB3Xb1, showed a clear zone around colonies. The plasmid DNA of the first subclone, TB3Xb1, was partially digested with Hin dIII and ligated into the pUC118/ Hin dIII/C.I.A.P. E. coli JM109 was transformed with the ligation mixture by the Hanahan method. The second subclones of the TB3Xb1 were incubated on LB agar plates with tributyrin and ampicillin for 5 days at 37℃. A positive clone, TB3Xb1H2, showed a clear zone around the colony, and was selected as the acyl-degrading enzyme-producing clone and sequenced. Analysis of sequenced data and sequenced similarity searches were performed using the BLAST program of the National Center for Biotechnology Information (NCBI). Homology alignment was performed with the CLUSTALW program [41] using MacVector 6.5 software (Oxford Molecular Group). The three-dimensional structure of CEST was p redicted u sing the PHYRE2 server ( http://www.sbg. bio.ic.ac.uk/phyre2 ) [22] .
- Expression and Purification of CEST
The carbohydrate esterase from M. thermotolerans was expressed in a heterologous system in E. coli . The gene encoding a putative carbohydrate esterase (CEST) was amplified with PCR using two primers that define the N-terminal without a signal peptide and C-terminal regions of the gene. The forward primer, TB3-CESTSP, was used in the amplification with an Eco RI site (italics and underline in the sequence): 5’-AGCACAGGA GAATTC GCTACC GAAGGCAAT-3’. The reverse primer, TB3-CEST-R, was used with a Sal I site (italics and underline in the sequence): 5’-AGCGCACATATC GTCGAC TTATTTACCGCA-3’. The reaction was performed in a TaKaRa PCR thermal cycler (TaKaRa, Japan). The PCR product was double-digested by Eco RI and Sal I and cloned 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 at 600 nm reached 0.4-0.5, 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added, followed by incubation for 24 h at 15℃. The cells were harvested by centrifugation at 6,000 rpm for 15 min at 4℃, and then suspended with binding buffer (20 mM sodium phosphate (pH 8.0), 0.5 M NaCl, and 5 mM imidazole). The cells were disrupted by sonication (pulse-on 30 sec, pulse-off 30 sec, 5 times, on ice), and the supernatant was collected by centrifugation at 13,000 rpm for 30 min at 4℃. The clear supernatant was loaded on to a HisTrap HP column (Amersharm Bioscience) equilibrated with binding buffer and eluted with elution buffer (20 mM sodium phosphate (pH 8.0), 0.5 M NaCl, and 0.5 M imidazole) at the flow rate of 1 ml/min. The eluted fractions were dialyzed overnight against 20 mM sodium phosphate (pH 8.0) and concentrated 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] . The concentrated proteins were used for determining the enzyme characterizations.
- Enzyme Assay
The purified CEST activity was measured spectrophotometrically using p -nitrophenyl acetate ( p NPA) (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℃ for 15 min according to Winkler and Stuckman [45] with some modifications [16] . The change in absorbance was measured over time at 410 nm using an Ultrospec 2000 pro UV/visible spectrophotometer (Amersham Bioscience).
The effect of pH on the activity of CEST was measured over a pH 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-NaOH buffer (pH 8.6-10.6). The optimal temperature of the CEST was determined by measuring the enzyme activity at various temperatures (5-70℃) in 20 mM sodium phosphate buffer (pH 8.0). For the temperature stability assay, the enzyme was preincubated with various buffers at 4℃ without substrate for 30 min. The extreme thermostability was determined by preincubating the CEST in 20 mM sodium phosphate buffer (pH 8.0) at various temperatures (50-70℃) for various hours (1-6 h). The effects of potential inhibitors or activators on the enzyme were determined by the addition of various metal salts to the reaction mixture at a final concentration of 1, 5, or 10 mM, which was preincubated for 10 min at 4℃. The test ions were BaCl 2 , CaCl 2 , CsCl, CuCl 2 , FeCl 2 , HgCl 2 , KCl, LiCl, NaCl, NiCl 2 , MgCl 2 , MnCl 2 , ZnCl 2 , and EDTA. The remaining activity was assayed as described above. For determination of the K m and k cat values, the assays contained substrates at concentrations of 0.1-1 mM. Kinetic parameters were obtained from the Lineweaver-Burk plots against various substrate concentrations using SWIFT II Applications software (Amersham Bioscience).
- Site-Directed Mutagenesis
Site-directed mutagenesis was carried out using a QuikChange XL site-directed mutagenesis kit (Stratagene), according to the manufacturer’s instructions. Primers used in the site-directed mutagenesis study are presented in Table 1 .
Primers used for site-directed mutagenesis.
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Primers used for site-directed mutagenesis.
- Nucleotide Sequence Accession Numbers
The nucleotide sequences of the 16S rDNA and carbohydrate esterase gene reported in this article were assigned as GenBank accession numbers KC571186 and KC571187.
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℃. 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 .
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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.
- 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-tributylinchloramphenicol plates. Some of these transformants were named as TB1-TB9. A selected clone, TB3, was partially and sequentially digested with Xba I and Hin dIII to obtain full nucleotide sequences of the tributylin-hydrolyzing enzyme from M. thermotolerans DAU221. The TB3 fragment, which was obtained after digestion with Xba I and Hin dIII, 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. 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 ).
BLASTP results of each ORF from the TB3/XbaI/HindIII fragment in GenBank.
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bp, base pairs; aa, amino acids.
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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.
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 the -10 region were located 80 bp upstream from the initiation codon with 19 bp spacing. Thus, the CE gene from DAU221 i s 864 bp, and encodes a p rotein 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.
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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.
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 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).
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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.
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Ribbon diagram showing the secondary structure of CEST with rainbow coloring from the amino-terminus (blue) to the carboxyl-terminus (red).
- 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 ).
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SDS-PAGE analysis of CEST. Lane 1: molecular weight marker. Lane 2: cell-free extract. Lane 3: purified CEST.
- 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℃, with p -nitrophenyl acetate as the substrate. The optimum pH was found to be 8.0 ( Fig. 7 A). 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℃), with the maximum activity observed at 15℃. On ice, this enzyme maintained 89% of its maximal activity (at 15℃) ( Fig. 7 B). Some of the cold-adapted carbohydrate esterases were described previously. These were pH 8.0 and 20℃ for cold-adapted PSHAa from Pseudoalteromonas halohplanktis TA125 [4] , 7.5 and 25℃ for EstO enzyme from Pseudoalteromonas arctica [2] , 7.5 and 25℃ for AELH from Acinetobacter sp. strain no. 6 [39] , and 8.0 and 15-20℃ for OLEI01171 from Oleispira antarctica [26] . These cold-adapted CEs showed 30-90% decrease in their activity at 5℃. However, CEST showed 1.5% decrease in its maximal activity at 5℃ and 11% decrease on ice. The activity of CEST was slightly reduced at 20℃, and it became insignificant at 60℃. CEST exhibited thermostability within a broad range of temperatures from 10℃ to 40℃. The preincubated CEST at 25℃ for 30 min showed its maximal activity (3.20 μmol/mg/min) and maintained 40-50% activity at 50-60℃ for 30 min. Furthermore, CEST was preincubated for 1, 3, or 6 h at 50℃, 60℃, or 70℃ for each incubation time ( Fig. 7 C). 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℃, 60℃, or 70℃. At 50℃ and 60℃, the results indicated that the preincubation of CEST for 6 h helped maintain 70% or more of its maximal activity. At 70℃, 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.
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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 50℃ (black triangle), 60℃ (black quardangle), or 70℃ (black circle).
To clarify the effect of various metal ions and reagents on CEST activity, the enzyme assays were carried out in the presence of Ba 2+ , Ca 2+ , Co 2+ , Cs 2+ , Cu 2+ , Fe 3+ , Hg 2+ , K + , Li 2+ , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Zn 2+ , or EDTA at final concentrationsof 1, 5, or 10 mM ( Table 3 ). When Na + , Mg 2+ , 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 Zn 2+ 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 Ba 2+ , Ca 2+ , Cs 2+ , Fe 3+ , K + , Li + , Mn 2+ , and Ni 2+ at 1, 5, or 10 mM, respectively. In particular, Cu 2+ and Hg 2+ 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 B2 T 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 Zn 2+ , whereas the presence of Ca 2+ led to mild a ctivation [5] . A PsyEst from Psychrobacter sp. Ant300 was inhibited by Mg 2+ and Mn 2+ , whereas its activity was enhanced in the presence of Ca 2+ [24] . The activity of PSHAa1385 from P. haloplanktis TAC125 has been reported to increase in the presence of Ca 2+ , Mg 2+ , and Mn 2+ [4] . The activity of Axe6A from Fibrobacter succinogenes decreased by more than 50% in the presence of Fe 2+ , Cu 2+ , and Zn 2+ at a concentration of 1 mM, but was unaffected by Mn 2+ , Co 2+ , Ca 2+ , Mg 2+ , and EDTA at a concentration of 10 mM. In contrast, the activity of Axe6B was inhibited by all ions, except Ca 2+ , at 10 mM [21] . To understand the basic catalytic parameters of CEST, steadystate kinetic analysis was performed. CEST had K m and k cat values of 0.278 mM and 1.9 s -1 , respectively, when p NPA was used as the substrate.
Effects of various metal ions on the activity of CEST.
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Data are shown as means ± standard errors. ND, not detected.
- Mutational Studies of CEST
CEST has three residues (Ser37, Glu192, and His281) that are highly conserved among the CE6 family proteins. Glutamate increases the pK a 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 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.
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Activity analysis of the site-directed mutants of CEST.
- 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.
Acknowledgements
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.
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