title: Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase.
abstract: An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.
authors.affiliation: Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan. a-nak@agbi.tsukuba.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Takakura
authors.forename: Naohisa
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Naoki
authors.initials: N
authors.lastname: Takaya
authors.forename: Kentaro
authors.initials: K
authors.lastname: Shiraki
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hoshino
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000235:genetics*; D011489:Protein Denaturation / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase