Enzymatic Analysis of a Thermostabilized Mutant of an Escherichia coli Hygromycin B Phosphotransferase
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概要
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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 °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 °C of HPH to 53 and 58.8 °C respectively. Specific activities and steady-state kinetics measured at 25 °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.
- 社団法人 日本農芸化学会の論文
著者
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Nakamura Akira
Division of Cardiology, Niigata University Graduate School of Medical & Dental Sciences
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Nakamura A
Fuji Oil Co. Ltd. Jpn
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Takaya Naoki
Division Of Applied Biochemistry University Of Tsukuba
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Hoshino Takayuki
Division Of Integrative Environmental Sciences Graduate School Of Life And Environmental Sciences Un
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Takakura Yasuaki
Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences,
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SUGIMOTO Naohisa
Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences,
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SHIRAKI Kentaro
Division of Applied Physics, Graduate School of Pure and Applied Sciences, University of Tsukuba
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