Stabilities of Chimeras of Hyperthermophilic and Mesophilic Glycerol Kinases Constructed by DNA Shuffling

概要

論文の詳細を見る
Glycerol kinases from Thermococcus kodakaraensis KOD1(Tk-GK)and Escherichia coli(Ec-GK)greatly differ in thermostability. The temperature(T_<1/2>)at which the enzymes lose half of their activity upon incubation for 20 min is 50-55℃ for Ec-GK and 〜95℃ for Tk-GK. To examine whether the amino acid substitutions that make Tk-GK more stable than Ec-GK are localized in a limited region, the chimeras of two parental genes encoding Tk-GK and Ec-GK were constructed by DNA shuffling. E.coli cells were transformed with a plasmid library harboring these chimeras and screened for those that produce chimeric enzymes which are more stable than Ec-GK. Four chimeric enzymes were isolated and purified, and their biochemical properties characterized. Replacement of 83 or 93 residues in the C-terminus of Ec-GK with the corresponding ones of Tk-GK increased the T_<1/2> value of Ec-GK by 25-30℃. In contrast, replacement of 85 residues in the N-terminus of Ec-GK with the corresponding ones of Tk-GK reduced the T_<1/2> value by 5-10℃. In addition, replacement of 10 residues in the C-terminus of Tk-GK with the corresponding ones of Ec-GK reduced the T_<1/2> value of Tk-GK by 〜15℃. Measurement of the far-UV CD spectra indicates that the three-dimensional structures of the chimeric enzymes, as well as those of the parent enzymes, are similar to one another. These results suggest that the amino acid substitutions responsible for the high stability of Tk-GK are largely localized in the C-terminal region.
社団法人日本生物工学会の論文
2001-06-25

Stabilities of Chimeras of Hyperthermophilic and Mesophilic Glycerol Kinases Constructed by DNA Shuffling

スポンサーリンク

概要

著者

関連論文

スポンサーリンク