title: Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli.
abstract: A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45 degrees C, respectively, and the enzyme maintained high activity at pH 5.0 and 60 degrees C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k(cat)/K(M)) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached approximately 71% at 20 degrees C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for alpha-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Yi-Shan
authors.initials: YS
authors.lastname: Chen
authors.forename: Guan-Chiun
authors.initials: GC
authors.lastname: Lee
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D019603:Thermoplasmales / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D008320:Maltose; D005964:Glucosyltransferases; C099692:trehalose synthase