Cell-based high throughput screening of a-amino acid ester hydrolase variants

Abstract:

[Objective] The study aimed to develop an efficient and sensitive high-throughput method to obtain alpha-amino acid ester hydrolase (AEH) with improved activity or thermo-stability. [Methods] Standard curve was made based on the fact that hydrolysis of cefaclor in alkaline buffer yields a derivate which has specific absorbance at 340 nm. Whole cell-based ultra-violet spectrophotometric method was applied to screen the cefaclor synthesis activity of AEH variants at a high-throughput scale. [Results] Beer’s Law is obeyed in the range of (0.1?0.6)×10?3 mol/L cefaclor. The average recovery is 99.8%?101.3%. 2 300 Clones obtained by one round of site-directed saturated mutagenesis were screened by this method. Three variants with more than 1.4-fold kcat and 4 variants with T50 5 °C more than wild type were ob-tained. [Conclusion] The screening method was precise and reliable. The screen capacity can be up to 2 000 samples per day, which was in the scale of high-throughput screening.

Key words:Alpha-amino acid ester hydrolase, Cefaclor, Ultra-violet spectrophotometry, Cell-based high throughput screening, Directed evolution