Directed Evolution System Of Bacillus Fastidiosus Uricase And Its Application

For directed evolution of Bacillus fastidiosus uricase, Mutants were from three libraries(A1-V150; V150-D212; Q160-L322) generated by error-prone PCR. The mutation rate was raised to 4.4% with final 1.5 mM MnCl2. Final 2.0 mM EDTA-Na2 was used to alleviate the effects of Mn2+ on ligation efficiency.After induced expression in 48-well microplates, transformed Escherichia coli cells were lyzed by 1.0 M Tris-HCl buffer at pH 9.0 plus 0.1% tween-20 and 1.0 mM 4-aminobenzamidine in 96-well microplates at 25 °C for 7.5~10.5 h; uricase reaction was monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm, to estimate Vm/Km by kinetic analysis of reaction curve. Uricase Vm/Km was resistant to initial uric acid levels with an upper limit three fold over that of initial rates.To recognize the one of higher activity in a uricase pair whose ratio of specific activities was 1.8 or 3.3, the area-under-the-curve by receiver-operator-curve analysis was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was developed to recognize positive mutants of one-fold higher activity.Indeed, one mutant bearing four mutations and higher activity was discovered from the library. Hence, the proposed system was practical for HTS of uricase mutants.