头孢菌素C乙酰化酶高通量筛选方法的改进

A modified high-throughput screening method for cephalosporin C acylase

目的改进头孢菌素C(cephalosporin C,CPC)乙酰化酶高通量筛选方法,获得催化活性显著提高的突变体。方法基于对二甲氨基苯甲醛(p-dimethylaminobenzaldehyde,p-DAB)与7-氨基头孢烷酸(7-amino cephalosporanic acid,7-ACA)的复合物在405 nm有最大吸收值的原理,采用无菌96孔深孔板对重组菌进行低温低浓度诱导剂诱导表达,再以含溶菌酶和DNase的缓冲液进行菌体裂解,最后以96孔板进行底物的水解和反应终止,经p-DAB显色后,利用酶标仪进行产物的快速定量,以实现CPC乙酰化酶突变体的高通量筛选。结果利用改进的p-DAB比色法从先前建立的突变文库中筛选到2个在13℃下比酶活明显提高的突变子1A10和2G8,其比酶活分别为1.7和2.4 U/mg,较原酶的0.85 U/mg显著提高。在优化的筛选条件下,A405的变化能够准确反映酶活性的大小,确保了该方法筛选结果的可信度。结论改进的p-DAB比色法具有筛选快速,操作简便,筛选通量大及准确度高等优点,能够提高CPC乙酰化酶改造及筛选的效率,为低温CPC乙酰化酶的创制提供了技术支持。

Objective To modify a high-throughput screening method for cephalosporin C （CPC） acylase to obtain a mutant with higher catalytic activity. Methods Based on the principle that the compound of p-dimethylaminobenza- ldehyde （p-DAB） and 7-amino cephalosporanie acid （7-ACA） had the maximum absorption at 405 nm, the recombinant strains were induced and expressed at low temperature and low concentration using sterile deep 96-well plate, and lyzed with the buffer containing lysozyme and DNase, then the substrate hydrolysis and reaction termination were carried out on 96-well plate. The products were subjected to rapid quantitative analysis by ELISA after p-DAB colour development reaction to realize the high-throughput screening of CPC acetlase mutants. Results Two mutants, 1A10 and 2G8, were screened from mutant library of CPC acylase by the modified p-DAB colorimetry. The specific activities of the two mutants were 1.7 and 2. 4 U/rag at 13 ℃ respectivly, which were significantly higher than that of the original eaylase （0. 8 U/mg）. Under the optimized condition for screening, the change of Am reflected the enzyme activity accurately, which guaranteed the reliability of the method. Conclusion The modified p-DAB colorimetry was rapid, simple and of high throughput and high accuracy, which increased the efficacy of modifcation and screening of CPC acylase, and provided a technical support for the development of cold CPC acylase.