title: Molecular cloning and enzymatic characterization of cyclomaltodextrinase from hyperthermophilic archaeon Thermococcus sp. CL1.
abstract: Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for 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, with optimal activity at 85℃. The melting temperature of TcCD was determined to be 93oC by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed α-cyclodextrin (α-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one α-1,4-glycosidic linkage of the CD ring to produce the corresponding single 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 6(2)-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 production of isomaltooligosaccharides in the food industry.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
authors.forename: Jae-Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: In-Hwan
authors.initials: IH
authors.lastname: Kim
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.forename: Sung-Gyun
authors.initials: SG
authors.lastname: Kang
authors.forename: James F
authors.initials: JF
authors.lastname: Holden
authors.forename: Jaeho
authors.initials: J
authors.lastname: Cha
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*; D047391:alpha-Cyclodextrins / Q000378:metabolism
chemical_list: D047391:alpha-Cyclodextrins; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase