全细胞高通量筛选a-氨基酸酯水解酶突变体的方法

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

摘要:

【目的】建立高效敏感的高通量筛选方法，用于筛选头孢克洛合成活性提高或热稳定性提高的a-氨基酸酯水解酶。【方法】根据头孢克洛在碱性条件下水解生成的衍生物在340 nm处有特征吸收峰的原理，制作出标准曲线。采用全细胞96孔板紫外分光光度法高通量测定a-氨基酸酯水解酶突变体的头孢克洛合成活性。【结果】头孢克洛含量与△A340?405在(0.1?0.6)×10?3 mol/L浓度范围内有良好的线性关系, 服从朗伯-比尔定律, 平均回收率为99.8%?101.3%。一轮定点饱和突变产生的2 300个克隆经该方法的筛选, 获得3株kcat提高40%以上, 4株半失活温度较野生型提高5 °C以上的突变体酶。【结论】该方法准确可靠，每天筛选量可达到2 000个反应, 达到高通量筛选的要求。

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.