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]. August 2013 ⎪ Vol. 23⎪ No. 8 1061 Lee et al. 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