Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.
|Number of pages||13|
|Journal||Applied Biochemistry and Biotechnology|
|State||Published - Jun 1 2016|
Bibliographical notePublisher Copyright:
© 2016, Springer Science+Business Media New York.
- Alkaline lysis
- Directed evolution
- High-throughput screening
- Positive mutants
- Pseudomonas aeruginosa arylsulfatase
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Molecular Biology