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

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Title
Molecular cloning and characterization of trehalose synthase from Thermotoga maritima DSM3109: Syntheses of trehalose disaccharide analogues and NDP-glucoses
Author(s)
S I Ryu; J E Kim; N T Huong; Eui-jeon Woo; S K Moon; S B Lee
Bibliographic Citation
Enzyme and Microbial Technology, vol. 47, no. 6, pp. 249-256
Publication Year
2010
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.
Keyword
Disaccharide analogueHyperthermophileNucleoside diphosphate-glucoseThermotoga maritimaTrehaloseTrehalose synthase
ISSN
0141-0229
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.enzmictec.2010.07.017
Type
Article
Appears in Collections:
Division of Biomedical Research > Disease Target Structure Research Center > 1. Journal Articles
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