基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性

Improving L-Asparaginase Activity from Bacillus licheniformis by Directed Evolution

为提高地衣芽孢杆菌L-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K_(cat)/K_m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α-螺旋数减少、无规则卷曲有所增加,表明其刚性略有降低,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L-天冬酰胺酶活性。

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 K_（cat）/K_m. 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 enzymatic activity. The circular dichroism analysis showed that the mutant enzymes contained less α-helix and more random coil than the wild enzyme, indicating a slight decrease in rigidity and an increase in flexibility. This study indicates that directional evolution can effectively improve the L-asparaginase activity from B. licheniformis.