| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
The purpose of this project is to prepare of novel oxidase by metal-substitution of urease active site. We purified and characterized new urease from photosynthetic bacterium, Rhodobacter capsulatus.
Urease, which catalyzes the hydrolysis of urea to form ammonia and carbamate, has a nickel binuclear active site. It is similar to binuclear metal ion center in the active sites in oxidases, such as methane monooxidase, catechol oxidase, catelase, which has Fe, Cu, and Mn ions, respectively. We have prepared metal-substituted ureases, that is Fe(II), Cu(II), and Mn(II) substituted ureases. Oxidation of guaiacol with H_2O_2 was catalyzed with Cu-substituted urease, while it was not occurred with Fe(II), Mn(II), and Ni(II) ureases. Kinetic parameters of guaiacol oxidation by carbamate-bridged and non-bridged Cu ureases were obtained as K_m : 2.5X10^<-3>, 2.5X10^<-3> M ; V_<max> : 16X10^<-7>, 7.1X10^<-7> M min^<-1>, respectively. The results suggest that the carbamate- bridge at the active site of oxidase is not essential for oxidase activity and substrate-binding, but is effective for improvement of oxidase activity. This study is the first example of functional conversion of hydrolase into oxidase. We proposed in this project that the metal substitution of metalloenzyme is useful method for functional conversion of enzyme.
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