A Practical System for High-Throughput Screening of Mutants of Bacillus fastidiosus Uricase

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• 作者：Tao Feng ; Xiaolan Yang ; Deqiang Wang ; Xiaolei Hu…
• 关键词：Bacillus fastidiosus uricase ; Directed evolution ; High ; throughput screening ; Cell lysate ; Error ; prone PCR
• 刊名：Applied Biochemistry and Biotechnology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：181
• 期：2
• 页码：667-681
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Biotechnology; Biochemistry, general;
• 出版者：Springer US
• ISSN：1559-0291
• 卷排序：181

文摘

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl₂, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V_m/K_m by kinetic analysis of reaction curve for comparison. V_m/K_m was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.