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【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α-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α-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。
【Objective】 To establish an efficient and sensitive high-throughput screening method for screening α-amino acid ester hydrolases with improved cefaclor synthesis activity or improved thermostability. 【Method】 According to the principle that cefaclor has a characteristic absorption peak at 340 nm under the alkaline conditions, a standard curve was prepared. High-throughput determination of cefaclor activity of α-amino acid ester hydrolase mutants by whole-cell 96-well plate UV spectrophotometry. 【Result】 The results showed that there was a good linear relationship between the content of cefaclor and △ A340? 405 in the range of (0.1? 0.6) × 10? 3 mol / L, and the average recoveries were 99.8% -103.3% . A total of 2 300 clones generated by the site-directed saturation mutation were screened by this method. Three mutant enzymes with kcat increase of more than 40% were obtained, and four mutant inactivation temperatures increased by more than 5 ° C compared with the wild type. 【Conclusion】 The method is accurate and reliable, with a daily screening of up to 2,000 reactions, which meets the requirements of high-throughput screening.