Molecular cloning and characterization of trehalose synthase from Thermotoga maritima DSM3109: Syntheses of trehalose disaccharide analogues and NDP-glucoses

Soo In Ryu, Jeong Eun Kim, Nguyen Thi Huong, Eui Jeon Woo, Sung Kwon Moon, Soo Bok Lee

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A gene (ORF TM0392) encoding a putative trehalose synthase (TmTreT) in Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and a glutathione-sepharose affinity column chromatography. The purified enzyme existed exclusively as a monomer in a native state. The optimum pH and temperature for this enzyme were 6.0 and 65°C. The glutathione-S-transferase (GST)-fusion enzyme had greater thermostability than thrombin-treated free enzyme. TmTreT had diverse substrate specificities. The enzyme effectively created a free trehalose from several nucleoside diphosphate (NDP)-glucoses as a donor and glucose as an acceptor. Inversely, the enzyme was also capable of employing several NDPs such as UDP, ADP, GDP, and CDP with trehalose to produce corresponding NDP-glucoses. The enzyme was able to employ other monosaccharides, such as mannose and fructose, as acceptors to synthesize disaccharide analogues of trehalose. The mannose-containing analogue was not hydrolyzed by trehalase and the rat intestinal enzymes. Furthermore, the analogue showed a competitive inhibition to the intestinal disaccharidases with Ki values of approximately 0.8-1.6mM. The results suggest that the enzyme is an useful trehalose synthase that can regenerate NDP-glucoses from NDPs and produce the inhibitory trehalose analogues of indigestible disaccharides.

Original languageEnglish
Pages (from-to)249-256
Number of pages8
JournalEnzyme and Microbial Technology
Volume47
Issue number6
DOIs
Publication statusPublished - 2010 Nov 8

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Thermotoga maritima
Trehalose
Diphosphates
Disaccharides
Cloning
Molecular Cloning
Nucleosides
Glucose
Enzymes
Mannose
Trehalase
trehalose synthase
Disaccharidases
Cytidine Diphosphate
Enzyme inhibition
Administrative data processing
Affinity chromatography
Agarose Chromatography
Column chromatography
Fructose

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

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title = "Molecular cloning and characterization of trehalose synthase from Thermotoga maritima DSM3109: Syntheses of trehalose disaccharide analogues and NDP-glucoses",
abstract = "A gene (ORF TM0392) encoding a putative trehalose synthase (TmTreT) in Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and a glutathione-sepharose affinity column chromatography. The purified enzyme existed exclusively as a monomer in a native state. The optimum pH and temperature for this enzyme were 6.0 and 65°C. The glutathione-S-transferase (GST)-fusion enzyme had greater thermostability than thrombin-treated free enzyme. TmTreT had diverse substrate specificities. The enzyme effectively created a free trehalose from several nucleoside diphosphate (NDP)-glucoses as a donor and glucose as an acceptor. Inversely, the enzyme was also capable of employing several NDPs such as UDP, ADP, GDP, and CDP with trehalose to produce corresponding NDP-glucoses. The enzyme was able to employ other monosaccharides, such as mannose and fructose, as acceptors to synthesize disaccharide analogues of trehalose. The mannose-containing analogue was not hydrolyzed by trehalase and the rat intestinal enzymes. Furthermore, the analogue showed a competitive inhibition to the intestinal disaccharidases with Ki values of approximately 0.8-1.6mM. The results suggest that the enzyme is an useful trehalose synthase that can regenerate NDP-glucoses from NDPs and produce the inhibitory trehalose analogues of indigestible disaccharides.",
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Molecular cloning and characterization of trehalose synthase from Thermotoga maritima DSM3109 : Syntheses of trehalose disaccharide analogues and NDP-glucoses. / Ryu, Soo In; Kim, Jeong Eun; Huong, Nguyen Thi; Woo, Eui Jeon; Moon, Sung Kwon; Lee, Soo Bok.

In: Enzyme and Microbial Technology, Vol. 47, No. 6, 08.11.2010, p. 249-256.

Research output: Contribution to journalArticle

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AU - Ryu, Soo In

AU - Kim, Jeong Eun

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