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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.