Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca²⁺. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca²⁺-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca²⁺ environments.

中文翻译：

---

通过共有设计获得具有改善的蛋白质热稳定性和酶活性的分选酶突变体。

金黄色葡萄球菌分选酶A（SaSrtA）是一种将蛋白质锚定于革兰氏阳性细菌细胞表面的酶。在由SaSrtA进行的转肽反应中，含有N末端甘氨酸的蛋白质可以与另一具有C末端LPXTG基序的蛋白质（X为任何氨基酸）共价连接。由于分选酶反应也可以在体外进行，因此在生物技术中发现了许多应用。尽管分选酶介导的连接具有许多优势，但SaSrtA受其低酶活性和对Ca ^2+的依赖性的限制。在我们的研究中，我们评估了SaSrtA野生型的热力学稳定性，并发现该酶稳定。我们进行了共识分析，以进一步提高酶的稳定性，同时增强酶的活性。结果，我们发现了热力学上改进的，更活跃的和Ca ²⁺独立的突变体。我们设想这些新的变体可以在低Ca ²⁺环境下的共轭反应中应用。