基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性.PDF

8 2017, Vol.38, No.22 食品科学 ※生物工程 基于定向进化技术提高地衣芽孢杆菌 L-天冬酰胺酶活性 邵泽香，焦琳舒，陆兆新，别小妹，赵海珍，张 充，吕凤霞* （南京农业大学食品科技学院，江苏南京 210095 ） 摘  要： L 为提高地衣芽孢杆菌 -天冬酰胺酶活性，通过定向进化技术对其进行分子改造。经过两轮易错聚合酶链 式反应和一轮DNA shuffling ，从19 100多个突变株中筛选到突变体S10、S16和S21，其酶比活力较野生型分别提高 了106%、74%和43%，且突变酶Kcat/Km 都有所增大。其中，突变体S10氨基酸序列发生3 个突变，K43E 、N67S和 I269L 。三维模拟结果显示，第43位氨基酸突变为谷氨酸、第67位氨基酸突变为丝氨酸，可能提高了底物亲和力和 α 催化效率，从而提高酶活性。圆二色谱分析表明，相比野生酶，突变酶的 -螺旋数减少、无规则卷曲有所增加，表 L 明其刚性略有降低，柔性有所增加。利用定向进化策略能够有效地提高地衣芽孢杆菌 -天冬酰胺酶活性。 L 关键词： -天冬酰胺酶；定向进化；酶活性 Improving L-Asparaginase Activity from Bacillus licheniformis by Directed Evolution SHAO Zexiang, JIAO Linshu, LU Zhaoxin, BIE Xiaomei, ZHAO Haizhen, ZHANG Chong, LÜ Fengxia* (College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China) Abstract: In order to improve its L-asparaginase activity, the L-asparaginase gene of Bacillus licheniformis was molecularly modified by directed evolution. Mutants S10, S16 and S21 were screened out of more than 19 100 mutants by two rounds of error-prone PCR and one round of DNA shuffling, whose specific activities were increased by 106%, 74% and 43%, respectively, as compared to that of the wild type, together increased Kcat/Km . The amino acid sequence of S10 showed three mutations, K43E, N67S and I269L. The results of three-dimensional simulation showed that amino acid mutations at position 43 for glutamic acid and at position 67 for serine may improve substrate affinity and catalytic efficiency, thereby increasing