title: Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4.
abstract: A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His6-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70-75 °C and 11.0, respectively. The kinetic parameters K m and V max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min(-1) for L-alanine, and 9.95 mM and 702.6 μmol min(-1) for D-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.
authors.affiliation: College of Life Science, Hebei Normal University, Shijiazhuang 050024, China.
authors.forename: Zhangwei
authors.initials: Z
authors.lastname: Xue
authors.forename: Yi
authors.initials: Y
authors.lastname: Hu
authors.forename: Shujing
authors.initials: S
authors.lastname: Xu
authors.forename: Kouhei
authors.initials: K
authors.lastname: Ohnishi
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.forename: Jiansong
authors.initials: J
authors.lastname: Ju
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
mesh_terms: D000411:Alanine Racemase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D000411:Alanine Racemase