title: Cloning, expression and characterization of a trehalose synthase gene from Rhodococcus opacus.
abstract: Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k cat/K m. Both 1 and 10 mM of Hg(2+), Cu(2+) and Al(3+) could inhibit the TreS activity, while only 1 mM of Ca(2+) and Mn(2+) could increase its activity. Five amino acid residues, Asp(244), Glu(286), Asp(354), His(147) and His(353), were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.
authors.affiliation: Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Main Street South, Zhongguancun, Beijing, 100081, China.
authors.forename: Junyan
authors.initials: J
authors.lastname: Yan
authors.forename: Yu
authors.initials: Y
authors.lastname: Qiao
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.forename: Hongbiao
authors.initials: H
authors.lastname: Ding
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005964:Glucosyltransferases; C099692:trehalose synthase