Molecular Cloning and Enzymatic Characterization of Cyclomaltodextrinase from Hyperthermophilic Archaeon Thermococcus sp. CL1
2013
Lee, J.E., Kyung Hee University, Yongin, Republic of Korea | Kim, I.H., Kyung Hee University, Yongin, Republic of Korea | Jung, J.H., Kyung Hee University, Yongin, Republic of Korea | Seo, D.H., Kyung Hee University, Yongin, Republic of Korea | Kang, S.G., Korea Ocean Research and Development Institute, Ansan, Republic of Korea | Holden, J.F., University of Massachusetts, Amherst, USA | Cha, J.H., Pusan National University, Busan, Republic of Korea
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 85oC. 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 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 production of isomaltooligosaccharides in the food industry.
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