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

Tao Feng, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, Juan Liao, Jun Pu, Xinyun Zhao, Chang Guo Zhan, Fei Liao

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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 Vm/Km by kinetic analysis of reaction curve for comparison. Vm/Km 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.

Original languageEnglish
Pages (from-to)667-681
Number of pages15
JournalApplied Biochemistry and Biotechnology
Volume181
Issue number2
DOIs
StatePublished - Feb 1 2017

Bibliographical note

Funding Information:
This work was supported by National Natural Science Foundation of China (nos. 30672139, 31570862 and 21402108), the Sciences and Technology Commission of Yuzhong District of Chongqing (no. 20130135), the Education Ministry of China (no. 20125503110007), and Chongqing Sciences and Technology Commission (CSTC2011BA5039). Some mutants reported here were claimed for invention patent in China (application no. 201610093491.8).

Publisher Copyright:
© 2016, Springer Science+Business Media New York.

Keywords

  • Bacillus fastidiosus uricase
  • Cell lysate
  • Directed evolution
  • Error-prone PCR
  • High-throughput screening

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

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