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Cloning, expression, properties, and functional amino acid residues of new trehalose synthase from Thermomonospora curvata DSM 43183
- Liang, Jiayuan, Huang, Ribo, Huang, Ying, Wang, Xiaobo, Du, Liqin, Wei, Yutuo
- Journal of Molecular Catalysis. B, Enzymatic 2013 v.90 pp. 26-32
- Escherichia coli, Thermomonospora curvata, active sites, amino acids, biocatalysts, catalytic activity, crystal structure, genes, isomaltulose, maltose, models, pH, screening, site-directed mutagenesis, substrate specificity, sucrose, temperature, trehalose, trehalulose, yields
- A new trehalose synthase (TreS) gene from Thermomonospora curvata DSM 43183 was cloned and expressed in Escherichia coli XL10-Gold. The purified recombinant enzyme (TreS-T.C) could catalyze the reversible interconversion of maltose and trehalose of sucrose into trehalulose without other disaccharides including isomaltulose at an optimum temperature of 35°C and a pH of 6.5. The Km of TreS-T.C for maltose (96mM) was lower than those for trehalose (198mM) and sucrose (164mM), suggesting that maltose is the optimum substrate. The maximum trehalose and trehalulose yields were 70% and >80%, respectively. Active TreS-T.C is a trimer comprising three identical 60kDa subunits. Homology modeling analysis revealed that TreS-T.C had a GH13-typical (β/α)8 barrel catalytic domain. Two sites, one determining substrate specificity (L116) and the other affecting product formation (E330), were found near the active center by homology modeling combined with site-directed mutagenesis. TreS-T.C may be used effectively as a potential biocatalyst for the production of trehalose and trehalulose from maltose and sucrose in a one-step reaction, respectively. This study also provides a feasible and effective method for studying functional amino acid residues around TreS without performing crystal structure analysis and high-throughput screening.