J. Microbiol. Biotechnol. (2013), 23(8), 1060–1069 http://dx.doi.org/10.4014/jmb.1302.02073 jmb

Molecular Cloning and Enzymatic Characterization of Cyclomalto- dextrinase from Hyperthermophilic Archaeon Thermococcus sp. CL1 Jae-Eun Lee1, In-Hwan Kim1, Jong-Hyun Jung1, Dong-Ho Seo1, Sung-Gyun Kang2, James F. Holden3, Jaeho Cha4, and Cheon-Seok Park1*

1Graduate School of Biotechnology and Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea 2Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea 3Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA 4Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea

Received: March 4, 2013 Revised: April 12, 2013 Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for Accepted: April 13, 2013 four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, o o First published online with optimal activity at 85 C. The melting temperature of TcCD was determined to be 93 C by June 3, 2013 both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion

*Corresponding author chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially Phone: +82-31-201-2631; hydrolyzed α-cyclodextrin (α-CD), and at the initial stage catalyzed a ring-opening reaction Fax: +82-31-204-8116; by cleaving one α-1,4-glycosidic linkage of the CD ring to produce the corresponding single E-mail: [email protected] maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-α-CD, G1-β- CD, and G2-β-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and 62-α-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the pISSN 1017-7825, eISSN 1738-8872 production of isomaltooligosaccharides in the food industry.

Copyright© 2013 by The Korean Society for Microbiology Keywords: Cyclomaltodextrinase, isomaltooligosaccharides, panose, Thermococcus and Biotechnology

Introduction starch and cellulose that are presumably located outside of the cells [13, 21, 28]. In accordance with these studies, the Like all living organisms, microorganisms require existence of a variety of α-glucan–metabolizing enzymes, carbon sources for their growth because carbon plays a amylopullulanase (APase), 4-α-glucanotransferase (4-α- fundamental role in the structure of all cellular molecules. GTase), and maltodextrin phosphorylase (MPase), was Hyperthermophilic bacteria and archaea are generally revealed and their possible roles in α-glucan assimilation found in high-temperature and anaerobic environments. were proposed [13]. It is suggested that 4-α-GTase produces Although most of these microorganisms exhibit a glucose from maltodextrins and maltose, whereas MPase chemolithoautotrophic mode of nutrition, some of them are converts maltodextrins to glucose-1-phosphate. In addition, designated as heterotrophs or opportunistic heterotrophs APase is an extracellular enzyme that was proven to be the [26]. Recently, α- and β-glucan utilization pathways and the major enzyme responsible for starch degradation. In presence of related genes were investigated in hyperthermophilic addition to these enzymes, cyclomaltodextrin glucanotransferase archaea Pyrococcus and Thermococcus species [21]. It has (CGTase) and cyclomaltodextrinase (CDase) might be been suggested that these species can utilize not only small involved in starch and maltose metabolism in Pyrococcus sugars such as maltose, but also polysaccharides including furiosus [13].

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Cyclomaltodextrinase (CDase, E.C. 3.2.1.54) is a member Materials and Methods of the glycoside hydrolase family 13 (GH13), and catalyzes the degradation of cyclomaltodextrins (or cyclodextrins, Bacterial Strains and Culture Conditions CDs) to maltose and glucose [23]. Recently, CDase, maltogenic Thermococcus sp. CL1 was isolated from a Paralvinella sp. amylases (MAase, E.C. 3.2.1.33), and neopullulanases polychaete worm collected from an active deep-sea hydrothermal (NPase, E.C. 3.2.1.35) have been categorized into a common vent sulfide chimney [2, 4]. Escherichia coli BL21-CodonPlus(DE3)- - - - r subfamily within GH13 [12] and are broadly called CD- RP [F ompT hsdS(rB mB ) dcm+ Tet galλ(DE3) endA Hte(argU proL Camr)] and DH10B [F– araD139 ∆(ara leu)7697 ∆lacX74 galU galK hydrolyzing enzymes [23]. These three enzymes are almost rpsL deoR Φ80lacZ∆M15 endA1 nupG recA1 mcrA ∆(mrr hsdRMS equivalent in terms of their catalytic properties and three- mcrBC)] were used as hosts for protein expression and cloning, dimensional structures [12]. They not only show hydrolyzing respectively. E. coli transformants were grown in Luria-Bertani activity on CDs and other carbohydrate substrates, (LB) medium (1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, including pullulan and soluble starch, but also perform and 0.5% (w/v) NaCl), containing ampicillin (100 µg/ml) and transglycosylation reactions using various biological chloramphenicol (34 µg/ml), at 37oC. Plasmids pGEM-T-easy vector materials as acceptors [17, 22, 30]. In biological systems, (Promega, USA) and pET-21a(+) (Novagen, USA) were used as CDase is suggested to be involved in the degradation of cloning and expression vectors, respectively. maltodextrin and glycogen in Bacillus subtilis, together with pullulanase [25]. Chemicals and Enzymes Although many studies have been carried out with α-Cyclodextrin (CD), γ-CD, acarbose, pullulan, maltooligosaccharides CDases from bacterial sources, few CDase-related enzymes (G2-G7), 6-O-α-D-glucosyl-α-CD (G1-α-CD), 6-O-α-D-glucosyl-β- CD (G1-β-CD), and 6-O-α-D-maltosyl-β-CD (G2-β-CD) were have been reported in hyperthermophilic archaea including purchased from Wako Pure Chemical Industries, Ltd. (Japan) and Staphylothermus marinus [16] and Thermofilum pendens [18]. β-CD, potato amylose, amylopectin, and ρ-nitrophenyl-α-D- Structural analysis of CDase from S. marinus revealed that maltohexaoside were purchased from Sigma Chemical Co. (St. it has a long N-terminal extension not found in other Louis, MO, USA). All restriction endonucleases were obtained bacterial counterparts. This extra N-terminal domain forms from New England Biolabs, Inc. (USA), and T4 DNA ligase was part of the substrate-binding pocket that resembles the purchased from Promega. Ex Taq DNA polymerase was obtained dimeric N-domain position in bacterial CDases [6]. from Takara Bio, Inc. (Japan). A recombinant CDase from E. coli Hashimoto et al. [1] suggested that CDase and CGTase play was purified using a Ni-NTA Superflow column (Qiagen, Germany). an important role in the starch metabolic pathway in Thermococcus sp. B1001. A novel starch degradation Cloning and Expression of the Gene Encoding CDase from pathway involving the extracellular conversion of starch Thermococcus sp. CL1 into CDs by CGTase and uptake of the CDs by a specific The gene encoding CDase from Thermococcus sp. CL1 (hereafter called tccd) was amplified by PCR with Ex Taq polymerase. The binding and transporting system following intracellular tccd-specific oligonucleotide primers flanking the 5’ and 3’ gene degradation by a CDase was proposed. Recently, we have ends were designed from the known tccd sequence. The forward sequenced the whole genome of Thermococcus sp. CL1. primer (TcCD-Nde, 5’-CAT ATG AAG GTG TAT AAA ATT TTC Together with recent advances in whole genome analysis, GGG TTC -3’) and the reverse primer (TcCD-Sal, 5’-GTC GAC our study revealed that CDases are somewhat prevalent in GCT GGT GTT CGG GGA GTA ATT TTT C -3’) contained NdeI Thermococcus species as in many bacterial strains. This and SalI restriction sites (underlined), respectively. The amplified indicates that CDase performs a significant role in the DNA fragments were cloned into the pGEM-T-easy vector and carbohydrate metabolism of this hyperthermophilic archaea, sequenced to confirm the full open reading frame of the tccd gene. similar to their bacterial counterparts. The confirmed error-free amplified fragment (1,935 bp) was Therefore, we analyzed and compared the gene digested with NdeI and SalI and then inserted into the expression organization near CDase in the whole genomes of four vector pET-21a(+) at the corresponding restriction sites. archaea strains, including Thermococcus sp. CL1, Pyrococcus Analysis of TcCD Sequence furiosus, Staphylothermus marinus, and Thermofilum pendens. Sequence homology analysis was performed using BLAST on In addition, the gene corresponding to CDase from the NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple Thermococcus sp. CL1 was cloned and expressed in Escherichia sequence alignments were performed by ClustalW2 [9]. The coli, and the enzymatic properties and hydrolysis activity phylogenetic tree was constructed using the MEGA 4.0 program pattern of the recombinant CDase were thoroughly based on the neighbor-joining method [27] and was evaluated by characterized. a bootstrap test on 1,000 replicates.

J. Microbiol. Biotechnol. Cyclomaltodextrinase from Thermococcus sp. CL1 1062

Purification of Recombinant TcCD Effects of Temperature, pH, and Other Chemicals on TcCD TcCD production by E. coli BL21-CodonPlus(DE3)-RP carrying Activity pET-tccd was induced by the addition of 0.5 mM isopropyl-β-D- The DNS method was used to determine the optimal thiogalactopyranoside at the mid-exponential growth phase and temperature and pH of TcCD. The dependence of enzyme activity incubation for 6 h at 37oC with agitation at 250 rpm. One liter of on pH was examined using sodium acetate (pH 4-5), sodium cultured cells was harvested by centrifugation (3,000 ×g for citrate (pH 5-6), sodium phosphate (pH 6-8), Tris-HCl (pH 7-9), 20 min at 4oC) and the pellet was washed with lysis buffer (50 mM and glycine-NaOH (pH 9-10) buffers. The optimal temperature

NaH2PO4, pH 7.5, 300 mM NaCl, and 10 mM imidazole). The was determined by examining TcCD activity in 40 mM sodium resuspended cells were disrupted by sonication (Sonifier 450; acetate buffer (pH 5.0) in a water bath at temperatures ranging Branson, USA; output 4, 6 times for 10s, constant duty) in an ice from 50oC to 98oC. The effects of metal ions (5 mM) and organic bath. The cell lysate was centrifuged at 12,000 ×g for 20 min at 4oC solvents (10%) were investigated by incubation at 80oC for 10 min. and then heated at 70oC for 20 min to remove all thermolabile E. Enzyme activity was determined after 1 h of preincubation at coli proteins. The crude enzyme was further purified using a Ni- room temperature using the DNS assay. NTA Superflow column (Qiagen) equilibrated with the same buffer. The column was washed with washing buffer [50 mM Differential Scanning Fluorimetry (DSF)

NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 7.5)] and DSF was performed as described previously [20]. A 7500 Fast

TcCD was eluted with elution buffer [50 mM NaH2PO4, 300 mM Real Time PCR System (Applied Biosystems, USA) was employed NaCl, and 250 mM imidazole (pH 7.5)]. The eluted fraction was for the heating cycle. Ten microliters each of enzyme (0.1 mg/ml) concentrated and dialyzed against 50 mM sodium acetate (pH 5.0) and a freshly prepared 10-fold water-based dilution of SYPRO to remove excess imidazole using a Centricon 10 filter from orange 5,000× (Invitrogen, USA) were added to a Fast Optical 96- Amicon (Millipore, USA). The molecular mass and the purity of Well reaction plate (Applied Biosystems) maintained on ice. The the TcCD were estimated by gel electrophoresis on a 10% (w/v) plate was sealed with optical adhesive film (Applied Biosystems) sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel. The and then gently vortexed for analysis in the real-time polymerase protein concentration was determined using the bicinchoninic acid chain reaction (RT-PCR) instrument. The heating cycle comprised (BCA) protein assay with bovine serum albumin as a standard. a gradient between 50oC and 99oC in 50 steps of 1 min, each with a 1oC ramp. Data were collected based on the calibration setting for

Enzyme Assay TAMRA dye detection (λex 560 nm; λem 582 nm) installed in the The relative and specific activities of TcCD for CDs (α-, β-, and instrument. γ-CD) and maltooligosaccharides (G2-G7) were assayed using two different methods. The hydrolytic activities of the enzyme Differential Scanning Calorimetry (DSC) were determined using dinitrosalicylic acid (DNS) as described by The melting temperature of TcCD was determined with the Miller [19] with slight modification. Fifty microliters of 1% (w/v) VP DSC (Microcal Inc., USA). Purified enzymes were dialyzed CD and 40 µl of 100 mM sodium acetate buffer (pH 5.0) were against 50 mM sodium acetate buffer (pH 5.0) and then concentrated mixed, and 10 µl of the enzyme was added to the solution. The to 1 mg/ml using a Centricon 10 filter (Millipore Co). The enzyme reaction mixture was incubated at 80oC for 5 min and the reaction samples and reference solution were degassed and carefully was terminated by the addition of 0.3 ml of DNS solution. After loaded into the cells to avoid bubble formation. After sealing, the boiling for 5 min, the absorbance of the reaction mixture was pans were heated from 20oC to 120oC at a rate of 1.5oC/min. The measured using a spectrophotometer (DU 730; Beckman, USA) at reference scan with buffer solution only preceded each sample 550 nm. One unit of enzyme activity was defined as the amount of run to achieve near-perfect baseline repeatability. enzyme that hydrolyzes 1 µmol equivalent of glycosidic bonds in the substrate in 1 min under the specified assay conditions (with Size-Exclusion Chromatography (SEC) maltose as the standard). A Superdex 200 GL column (10 × 300 mm, Amersham To examine the hydrolysis activity of TcCD for maltooligosaccharides Biosciences, Sweden) was used to estimate the apparent molecular (G2-G7), we used the glucose oxidase-peroxidase (GOD-POD) mass of TcCD. Protein samples (4 mg/ml) were applied to the method with 10 mM of each substrate. After incubation at 80oC for column system equilibrated in a 50 mM sodium phosphate buffer 5 min, the reaction was terminated by quenching on ice and 0.9 ml (pH 7.0) and eluted with the same buffer at a flow rate of 0.3 ml/min. of GOD solution (Sinyang Chem. Co., Korea) was added to Carbonic anhydrase (29 kDa), albumin (66 kDa), β-amylase (200 kDa) determine the glucose concentration in the reaction mixture. Color and thyroglobulin (669 kDa), were used as molecular standards for development was monitored at 505 nm using a spectrophotometer. the calibration to estimate the apparent molecular mass of the enzyme. One unit of enzyme activity was defined as the amount of enzyme Thin-Layer Chromatography (TLC) that hydrolyzes 1 µmol equivalent of glycosidic bonds in the The hydrolytic patterns of TcCD were determined using 0.5% substrate in 1 min under the reaction conditions. (w/v) α-, β-, and γ-CDs, pullulan, acarbose, amylose, amylopectin,

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G1-α-CD, G1-β-CD, G2-β-CD, and 10 mM maltooligosaccharides are closely related. However, recent whole genome (G2-G7) and ρ-nitrophenyl-α-D-maltohexaoside (ρNP6) in 40 mM research revealed that the genus Thermococcus has a slightly sodium acetate buffer (pH 5.0) at 80oC. The reaction products larger genome than the genus Pyrococcus, implying that were analyzed by silica gel K5F TLC (Whatman, UK), with there might be a differential gain of genes such as CDase isopropyl alcohol/ethyl acetate/water (3:1:1 (v/v/v)) as the during the evolution of Thermococcus after it branched off solvent system. After irrigating twice, each plate was dried and from the genus Pyrococcus. then visualized by immersion in a solution containing 0.3% (w/v) The most interesting finding is that the gene organization N-(1-naphthyl)-ethylenediamine and 5% (v/v) H SO in methanol, 2 4 near CDases is quite similar in Thermococcus and Pyrococcus followed by heating for 10 min at 110oC [24]. genera (Fig. 1A). Maltodextrin or sugar transport-related High-Performance Anion-Exchange Chromatography (HPAEC) genes and the amylopullulanase gene are located on one The hydrolysis products of branched cyclodextrins (G1-α-CD, side of the CDases. The orientation and location of these G1-β-CD, and G2-β-CD) were analyzed by HPAEC using a Dionex genes are the same in all Thermococcus and Pyrococcus model DX-600 system consisting of a GS50 gradient pump and genera. Unlike Euryarchaeota, it is not easy to find any ED50 electrochemical detector and PA-1 anion-exchange analytical significant correlation in gene arrangement close to CDases column (Dionex, USA). The separation gradient was composed of in Crenarchaeota. In Staphylothermus marinus, genes for eluent A (150 mM NaOH) and eluent B (150 mM NaOH in 600 transcriptional regulators, voltage-gated family proteins, mM sodium acetate solution), with a gradient profile of eluent B and ribosomal proteins are positioned near the CDase. (1.0 ml/min) at 35% for 30 min and 100% for 10 min. However, pyruvate ferredoxin/flavodoxin oxidoreductase,

rubredoxin-type Fe(Cys)4 protein, and DNA polymerase Results and Discussion are adjacent to CDase in Thermofilum pendens. This result indicates that CDase is widespread in most of the Genome Organization near CDase in Hyperthermophilic Thermococcus genus and may play an essential role in sugar Archaea metabolism in this branch of hyperthermophilic archaea. CDases most likely participate in the metabolism of small oligosaccharides and are important in the generation Sequence Analysis of the CDase Gene in Thermococcus of energy from various carbon sources in bacteria such as sp. CL1 B. subtilis and Klebsiella oxytoca [25]. Recently, several Recently, we completely sequenced the whole genome CDases and their homologs have been reported in of Thermococcus sp. CL1, a sulfur-reducing hyperthermophilic hyperthermophilic archaea including Staphylothermus marinus archaeon [4]. Analysis of the complete genome of Thermococcus and Thermofilum pendens [16, 18]. In addition, a BLAST sp. CL1 revealed the presence of an open reading frame, search revealed that CDases and their homologs are CL1_0884 (called tccd hereafter), encoding a protein sequence widespread in hyperthermophilic archaea (data not shown). homologous to previously reported CDases. CL1_0884 This indicates that CDases may play an important role in consists of 1,935 bp, corresponding to 644 amino acids, hyperthermophilic archaea, similar to their role in bacteria. with a theoretical molecular mass of 75,478.38 Da and pI of We analyzed and compared the genome organization 8.13. Multiple sequence alignment of the TcCD amino acid near CDases in four different hyperthermophilic archaea sequence revealed that TcCD had the highest sequence species, Pyrococcus, Staphylothermus, Thermococcus, and identity (70%) with a CDase from Thermococcus sp. 4557, Thermofilum (Fig. 1A). Pyrococcus and Thermococcus species followed by 63% sequence identity with Thermococcus are the most common hyperthermophilic archaea and belong onnurineus NA1. It also showed relative similarity with the to the Euryarchaeota phylum, whereas Staphylothermus and sequences of CDase of P. yayanosii CH1 (61%) and CD- Thermofilum species are members of the Crenarchaeota hydrolyzing enzyme from P. furiosus DSM 3638 (56%). In phylum. In Pyrococcus species, CDase homologs are found Crenarchaeota, 56% sequence identity was observed with in only two species, P. furiosus and P. yayanosii, among a the CDase homolog of Thermofilum pendens Hrk5 [18], total of eight species whose whole genome sequences have whereas comparatively low sequence identity (26%) was been completed and are available in the database [3, 5, 7, observed with MAase from S. marinus [16]. Fig. 1B shows 10]. However, all Thermococcus species (7 strains), except T. the phylogenetic analysis using amino acid sequences of sibiricus, possess CDases. Despite the fact that the genus archaeal CDases and their homologs. The sequence analysis Pyrococcus has a higher optimum growth temperature also showed that TcCD possesses four conserved regions (95oC-103oC) than Thermococcus (75oC-93oC), both genera (Fig. 1C) that are typical of CD-hydrolyzing enzymes,

J. Microbiol. Biotechnol. Cyclomaltodextrinase from Thermococcus sp. CL1 1064

Fig. 1. Phylogenetic analysis of the cyclomaltodextrinase genes and their surrounding genome organization in hyperthermophilic archaea. (A) Schematic presentation of the gene clusters for the cyclodextrin-hydrolyzing enzyme in Thermococcus sp. CL1, Pyrococccus furiosus, Thermofilum pendens, and Staphylothermus marinus. Arrows indicate the orientation of the coding sequences. (B) Phylogenetic tree based on amino acid sequences of various CDases in Thermococcales, Thermofilum pendens, and Staphylothermus marinus. Multiple alignment was carried out using ClusterW2 [9]. A phylogenetic tree was constructed using the neighbor-joining method of the MEGA program (ver 4.0) [27]. The number near the branch indicates bootstrap values based on 1,000 replicates. (C) Comparison of amino acid sequences in the conserved regions (I, II, III, IV) of cyclodextrin-hydrolyzing enzymes. The arrows indicate catalytic amino acid residues and black boxes indicate highly conserved amino acid residues. TcCD, cyclodextrinase from Thermococcus sp. CL1; B1001, cyclodextrinase from Thermococcus sp. B1001; TfMA, maltogenic amylase from Thermofilum pendens; PFTA, cyclodextrin-hydrolyzing enzyme from Pyrococcus furiosus; TPMA, maltogenic amylase from Thermoplasma volcanium GSS1; SMMA, maltogenic amylase from Staphylothermus marinus.

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including CDase, MAase, and NPase [1, 14, 16, 18, 29]. In addition, TcCD possesses an N-terminal extension composed of 193 amino acid residues found in MAase from S. marinus and CD-hydrolyzing enzyme of P. furiosus DSM 3638. The N-terminal extra domain of these enzymes is known to be composed of all of the substrate-binding components in a monomeric unit, which is generally provided by the dimeric N-domain position in bacterial enzymes [11, 12, 23]. These results indicate that TcCD belongs to the GH13 family of CD-hydrolyzing enzymes and retains CDase activity.

Cloning of the CL1_0884 Gene and Expression of Recombinant TcCD Recombinant TcCD was effectively expressed in E. coli with a six-histidine tag at its C-terminus. Most of the endogenous E. coli proteins in the crude extract were Fig. 2. SDS-PAGE of purified recombinant TcCD. eliminated by heat treatment at 70oC for 20 min, and TcCD Lane M, protein size marker; Lane 1, proteins from crude extract; could be isolated by Ni-NTA affinity chromatography. Lane 2, proteins after heat treatment (70oC, 20 min); Lane 3, purified SDS-PAGE analysis indicated that TcCD was purified to TcCD after Ni-NTA affinity chromatography. apparent homogeneity with a molecular mass of about 65 kDa, which was not in agreement with the expected determined by protein sequencing and exactly matched molecular mass of 75,956 Da (Fig. 2). However, there are with those deduced from the tccd gene (data not shown). quite a few examples of unusual mobility of expressed proteins in gels of SDS-PAGE, and fast movement in SDS- Thermostability of TcCD PAGE has previously been described for thermostable The optimal reaction temperature and pH of TcCD for proteins originating from hyperthermophilic archaea [15]. α-CD were 85oC and pH 5.0 (50 mM sodium acetate buffer), This result might be explained by the imperfect denaturation respectively (Fig. 3). As shown in Table 1, all metal ions of the proteins by the denaturing gel loading buffer. The inhibited TcCD activity at the concentration of 5 mM, first seven amino acids of purified TcCD (MYKIFGF) were except for KCl and NaCl. Dimethyl sulfoxide, methanol, and ethanol (10%) inhibited the catalytic activity of TcCD.

Fig. 3. Effects of pH (A) and temperature (B) on TcCD activity. The reactions were performed using 0.5% α-cyclodextrin as the substrate in 40 mM sodium acetate (pH 4-5), sodium citrate (pH 5-6), sodium phosphate (pH 6-8), Tris-HCl (pH 7-9), or glycine-NaOH (pH 9-10) buffer at 50oC-98oC. The activities were determined by the dinitrosalicylic acid method.

J. Microbiol. Biotechnol. Cyclomaltodextrinase from Thermococcus sp. CL1 1066

Table 1. Effects of different metal ions (5 mM) and organic solvents (10%) on TcCD activity. Reagents Relative activity (%) None 100 KCl 122 NaCl 106

CaCl2 94

MgCl2 79

CoCl2 71

BaCl2 50

NiCl2 39

AlCl3 18 TLC analysis of hydrolysis products from various CdCl2 3 Fig. 4. substrates incubated with 3.8 units of TcCD at 80oC and pH ZnCl2 0 5.0 for 24 h. CuCl2 0 S, G1-G7 standards; G2, maltose; G3, maltotriose; α-CD, α- FeSO 0 4 cyclodextrin; AL, amylose; AP, amylopectin; SS, soluble starch; Pul, FeCl 0 2 pullulan; Acar, acarbose. DMSO 39 MeOH 1 EtOH 0 were rarely detected. However, neither glucose nor maltose was detected in the reaction with panose as a substrate. The reactions were performed at 80oC for 10 min and activities were determined by the dinitrosalicylic acid method. This result indicated that TcCD possessed a weak hydrolysis activity toward α-1,4-glycosidic linkages in high molecular mass substrates, but not in small substrates such as panose. In thermal inactivation experiments, the half-life of TcCD The reaction pattern of TcCD on CDs revealed that the was 8.3 h at 90oC, indicating a highly thermostable characteristic, enzyme initially performed ring opening, followed by as expected (data not shown). The extremely thermostable hydrolysis of the resulting maltooligosaccharides (data not properties of the enzyme were confirmed by DSC and DSF. shown). The specific activities towards α-CD, β-CD, and γ- o DSC analysis gave a melting temperature (Tm) of 92.8 C, CD were 93.0, 24.7, and 18.4 U/mg, respectively, indicating o whereas DSF yielded a Tm of 93.0 C (data not shown). It has that α-CD was the most favorable substrate for TcCD. The been suggested that substrate preference and thermostability action pattern of TcCD on various maltooligosaccharides might be related to an oligomeric state of the enzyme, as (G2 – G7) was quite similar to one another (Fig. 5), except shown for maltogenic amylase from the hyperthermophilic for G2, which was not a substrate for TcCD. Although G2 archaeon Thermoplasma volcanium GSS1 [8]. However, this was not cleaved by TcCD, maltooligosaccharides longer is not the case for TcCD, which was shown to be in a than G2 were hydrolyzed to glucose and G2. The specific monomeric state by SEC with a Superdex 200 HR column activity towards maltooligosaccharides revealed that a (data not shown). long substrate (G7) is preferred over smaller substrates (G3-G6) (Fig. 5). Overall, the substrate preference of TcCD Action Patterns of TcCD was CD>>maltodextrins>soluble starch> pullulan. This is TcCD reacted with various substrates including CDs (α- somewhat different from the substrate preference of CD, β-CD, and γ-CD), maltooligosaccharides (G2-G7), amylose, MAase from S. marinus, which shows the highest activity amylopectin, and soluble starch to produce predominantly toward acarbose and pullulan, which were hydrolyzed to glucose and maltose, as shown for typical CD-hydrolyzing acarviosine-glucose and panose, respectively [16]. enzymes (Fig. 4). Pullulan was not a preferred substrate When branched CDs (G1-α-CD, G1-β-CD, and G2-β- since it was not degraded by 4.8 units of TcCD, which was CD) were reacted with TcCD, one major reaction product enough to hydrolyze soluble starch or amylopectin. However, other than glucose and G2 was observed in each reaction. the addition of excess TcCD could produce panose as a TLC and HPAEC analyses suggested that the product (P1) principal degradation product, although glucose and maltose appearing near G4 in both G1-α-CD and G1-β-CD reactions

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Fig. 5. TLC analysis of the action mode of 0.32 units of TcCD on various maltooligosaccharides from maltotriose to maltoheptaose (10 mM) for 0-60 min at 80oC. S, G1-G7 standards. was the same compound, whereas the product (P2) observed location of P1 in TLC and the elution time in HPAEC in the G2-β-CD reaction was different (Fig. 6). The density analysis were identical to those of panose, strongly of these spots increased as the reaction proceeded, indicating suggesting that P1 is panose. This suggests that P2 is 62-α- that TcCD could not react with these products further. The maltosylmaltose (Fig. 6C). When the reactions of α-CD and

Fig. 6. TLC analysis of hydrolysis products from 0.5% glucosyl-α-CD (A), glucosyl-β-CD (B), and maltosyl-β-CD (C) incubated with 38 units of TcCD at 80oC for various times (5-180 min). S, G1-G7 standards; P, panose.

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Table 2. Comparison of enzymatic properties of TcCD and other archaeal cyclodextrin-hydrolyzing enzymes. B1001 TfMA PFTA TPMA SMMA TcCD Homology (%) 54 52 53 24 25 100 Optimal 95 95 90 80 100 85 temperature (oC) Optimal pH 5.5 5.5 4.5 5.5 5.0 5.0 Oligomeric state N.D. N.D. Monomer/dimer Dimer Monomer Monomer Substrate CD>>MD>SS CD>>MD>PL=SS CD>>MD>PL=SS MD=CD>>PL=SS G6>CA>γ-CD CD>>MD>SS>PL preference Optimal β-CD γ-CD α-CD G5 G6 α-CD substrate Final hydrolysis G1, G2 G1, G2 G3, G4 G1, G2 G1, G2 G1, G2 product Reference [1] [18] [29] [8] [16] This study MD, maltodextrin; CA, cycloamylose; CD, cyclodextrin; PL, pullulan; SS, soluble starch. B1001, CDase from Thermococcus sp. strain B1001; TfMA, maltogenic amylase from Thermofilum pendens; PFTA, CD-hydrolyzing enzyme from P. furiosus; TPMA, maltogenic amylase from Thermoplasma volcanium GSS1; SMMA, maltogenic amylase from Staphylothermus marinus. N.D., not determined.

G1-α-CD with TcCD were compared, one other noticeable References difference in the reaction products was observed, in addition to panose. Whereas a significant amount of G3 appeared in 1. Hashimoto Y, Yamamoto T, Fujiwara S, Takagi M, Imanaka T. the α-CD reaction, only a trivial amount of G3 was produced 2001. Extracellular synthesis, specific recognition, and intracellular in the G1-α-CD reaction. This result suggested that branched degradation of cyclomaltodextrins by the hyperthermophilic glucose in G1-α-CD was not inhibited by the normal binding archaeon Thermococcus sp. strain B1001. J. Bacteriol. 183: of linearized G1-α-CD on the subsite in the enzyme that 5050-5057. 2. Holden JF, Takai K, Summit M, Bolton S, Zyskowski J, prevents the hydrolysis of α-1,6-glycosidic linkages and the Baross JA. 2001. Diversity among three novel groups of release of G3 from linearized G1-α-CD. A similar result hyperthermophilic deep-sea Thermococcus species from three was observed with G1-β-CD and G2-β-CD reactions, in sites in the northeastern Pacific Ocean. FEMS Microbiol. Ecol. 2 which panose and 6 -α-maltosylmaltose, respectively, were 36: 51-60. the major products other than glucose and G2. 3. Jun X, Lupeng L, Minjuan X, Oger P, Fengping W, Jebbar In conclusion, genomic-based analysis revealed that M, Xiang X. 2012. Complete genome sequence of the CDases are widespread in the hyperthermophilic archaea, obligate piezophilic hyperthermophilic archaeon Pyrococcus especially in the Thermococcus genus. Functional expression yayanosii CH1. J. Bacteriol. 193: 4297-4298. of CDase from Thermococcus sp. CL1 demonstrated that it 4. Jung JH, Holden JF, Seo DH, Park KH, Shin H, Ryu S, et al. displayed typical CDase-like activity, with small variations 2012. Complete genome sequence of the hyperthermophilic such as oligomeric state and substrate preference. Table 2 archaeon Thermococcus sp. strain CL1, isolated from a shows the comparison of various CDases and CDase-like Paralvinella sp. polychaete worm collected from a hydrothermal vent. J. Bacteriol. 194: 4769-4770. enzymes with TcCD. These results indicate that CDases are 5. Jung JH, Lee JH, Holden JF, Seo DH, Shin H, Kim HY, et al. prevalent in hyperthermophilic archaea and may play an 2012. Complete genome sequence of the hyperthermophilic important function in sugar metabolism, as in the bacterial archaeon Pyrococcus sp. strain ST04, isolated from a deep- kingdom. sea hydrothermal sulfide chimney on the Juan de Fuca Ridge. J. Bacteriol. 194: 4434-4435. Acknowledgments 6. Jung TY, Li D, Park JT, Yoon SM, Tran PL, Oh BH, et al. 2012. Association of novel domain in active site of archaic This work was supported by a National Research hyperthermophilic maltogenic amylase from Staphylothermus Foundation of Korea (NRF) grant funded by the Korean marinus. J. Biol. Chem. 287: 7979-7989. Government (MEST) (No. 2012-0005289). 7. Kawarabayasi Y, Sawada M, Horikawa H, Haikawa Y, Hino Y, Yamamoto S, et al. 1998. Complete sequence and gene

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organization of the genome of a hyper-thermophilic Thermofilum pendens with extremely thermostable maltogenic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5: 55- amylase activity. Appl. Microbiol. Biotechnol. 85: 1821-1830. 76. 19. Miller GL. 1959. Use of dinitrosalicylic acid reagent for 8. Kim JW, Kim YH, Lee HS, Yang SJ, Kim YW, Lee MH, et al. determination of reducing sugar. Anal. Chem. 31: 426-428. 2007. Molecular cloning and biochemical characterization of 20. Niesen FH, Berglund H, Vedadi M. 2007. The use of the first archaeal maltogenic amylase from the hyperthermophilic differential scanning fluorimetry to detect ligand interactions archaeon Thermoplasma volcanium GSS1. Biochim. Biophys. that promote protein stability. Nat. Protoc. 2: 2212-2221. Acta 1774: 661-669. 21. Oslowski DM, Jung JH, Seo DH, Park CS, Holden JF. 2011. 9. Larkin MA, Blackshields G, Brown NP, Chenna R, Production of hydrogen from α-1,4- and β-1,4-linked McGettigan PA, McWilliam H, et al. 2007. Clustal W and saccharides by marine hyperthermophilic Archaea. Appl. Clustal X version 2.0. Bioinformatics 23: 2947-2948. Environ. Microbiol. 77: 3169-3173. 10. Lee HS, Bae SS, Kim MS, Kwon KK, Kang SG, Lee JH. 2011. 22. Park KH, Kim MJ, Lee HS, Han NS, Kim D, Robyt JF. 1998. Complete genome sequence of hyperthermophilic Pyrococcus Transglycosylation reactions of Bacillus stearothermophilus sp. strain NA2, isolated from a deep-sea hydrothermal vent maltogenic amylase with acarbose and various acceptors. area. J. Bacteriol. 193: 3666-3667. Carbohydr. Res. 313: 235-246. 11. Lee HS, Kim JS, Shim K, Kim JW, Inouye K, Oneda H, et al. 23. Park KH, Kim TJ, Cheong TK, Kim JW, Oh BH, Svensson B. 2006. Dissociation/association properties of a dodecameric 2000. Structure, specificity and function of cyclomaltodextrinase, cyclomaltodextrinase. Effects of pH and salt concentration a multispecific enzyme of the α-amylase family. Biochim. on the oligomeric state. FEBS J. 273: 109-121. Biophys. Acta 1478: 165-185. 12. Lee HS, Kim MS, Cho HS, Kim JI, Kim TJ, Choi JH, et al. 24. Robyt JF, Mukerjea R. 1994. Separation and quantitative 2002. Cyclomaltodextrinase, neopullulanase, and maltogenic determination of nanogram quantities of maltodextrins and amylase are nearly indistinguishable from each other. J. Biol. isomaltodextrins by thin-layer chromatography. Carbohydr. Chem. 277: 21891-21897. Res. 251: 187-202. 13. Lee HS, Shockley KR, Schut GJ, Conners SB, Montero CI, 25. Shim JH, Park JT, Hong JS, Kim KW, Kim MJ, Auh JH, et al. Johnson MR, et al. 2006. Transcriptional and biochemical 2009. Role of maltogenic amylase and pullulanase in analysis of starch metabolism in the hyperthermophilic maltodextrin and glycogen metabolism of Bacillus subtilis archaeon Pyrococcus furiosus. J. Bacteriol. 188: 2115-2125. 168. J. Bacteriol. 191: 4835-4844. 14. Lee MH, Kim YW, Kim TJ, Park CS, Kim JW, Moon TW, et 26. Stetter KO. 2006. Hyperthermophiles in the history of life. al. 2002. A novel amylolytic enzyme from Thermotoga maritima, Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1837-1843. resembling cyclodextrinase and α-glucosidase, that liberates 27.Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: glucose from the reducing end of the substrates. Biochem. Molecular evolutionary genetics analysis (MEGA) software Biophys. Res. Commun. 295: 818-825. version 4.0. Mol. Biol. Evol. 24: 1596-1599. 15. Lee MH, Yang SJ, Kim JW, Lee HS, Park KH. 2007. 28. Xavier KB, Peist R, Kossmann M, Boos W, Santos H. 1999. Characterization of a thermostable cyclodextrin glucanotransferase Maltose metabolism in the hyperthermophilic archaeon from Pyrococcus furiosus DSM3638. Extremophiles 11: 537-541. Thermococcus litoralis: purification and characterization of key 16. Li D, Park JT, Li X, Kim S, Lee S, Shim JH, et al. 2010. enzymes. J. Bacteriol. 181: 3358-3367. Overexpression and characterization of an extremely thermostable 29. Yang SJ, Lee HS, Park CS, Kim YR, Moon TW, Park KH. maltogenic amylase, with an optimal temperature of 100oC, 2004. Enzymatic analysis of an amylolytic enzyme from the from the hyperthermophilic archaeon Staphylothermus marinus. hyperthermophilic archaeon Pyrococcus furiosus reveals its N. Biotechnol. 27: 300-307. novel catalytic properties as both an α-amylase and a 17. Li D, Park SH, Shim JH, Lee HS, Tang SY, Park CS, et al. cyclodextrin-hydrolyzing enzyme. Appl. Environ. Microbiol. 2004. In vitro enzymatic modification of puerarin to puerarin 70: 5988-5995. glycosides by maltogenic amylase. Carbohydr. Res. 339: 2789- 30. Yoon JW, Jeon EJ, Jung IH, Min MJ, Lee HY, Kim MJ, et al. 2797. 2003. Maltosyl-erythritol, a major transglycosylation product 18. Li X, Li D, Yin Y, Park KH. 2010. Characterization of a of erythritol by Bacillus stearothermophilus maltogenic amylase. recombinant amylolytic enzyme of hyperthermophilic archaeon Biosci. Biotechnol. Biochem. 67: 525-531.

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