定向进化改造猪肾氨基酰化酶Ⅰ底物特异性

Improvement of the Substrate Specificity and thermostability of Porcine Aminoacylase Ⅰ by Directed Evolution

应用定向进化技术提高了猪肾氨基酰化酶I(pACY1)对乙酰-L-精氨酸和乙酰-L-苯丙氨酸的底物特异性和热稳定性。结合易错PCR和DNA改组的方法,构建了pACY1的突变体文库;利用营养选择平板和96孔板耐热双重筛选体系。经过5轮改组和筛选,得到突变酶7E15对乙酰-L-精氨酸和乙酰-L-苯丙氨酸的特异性活力分别提高了23倍和9倍,Tm值提高了7℃。突变酶的氨基酸序列中有7个氨基酸残基发生了替换,分别是L176A、R196T、A283G、V304G、K306Q、V369A和L370T。结构模拟结果显示,突变位点L176A、V369A和L370T靠近酶活性中心,影响了底物的结合;而R196T、A283G、V304G和K306Q离酶活性中心较远,可能对酶的热稳定性起到了关键作用。

Directed evolution was used to improve the specificity and thermostability of porcine aminoacylase I （pACY1） hydrolyzing N-acetyl-L-Arg and N-acetyl-L-Phe. A mutant libarary was constructed by error-prone PCR and DNA shuffling. The positive clones were screened by nutrition selective-culture plates and 96-well plates ther- moresistance. After 5 cycle shuffling and screening, One mutant named 7E15 was selected. It exhibited 23-fold in- creased activity and 7~C increased Tm by hydrolyzing N-acetyl-L-Arg and 9-fold increased activity by hydrolyzing N-acetyl-L-Phe comparing its wild type parent. Sequence analysis revealed that 7E15 has 7 amino acid substitu- tions-namely L176A, R196T, A283G, V304G, K306Q, V369A and L370T. L176A, V369A and L370T are near to the activity center in the model of pACYI, and they were suggested to bind with the substrates; while the other 4 were distant, may play key roles in the change of the thermostability of pACYI.