College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Yantai Institute of China Agricultural University, Yantai 264670, China.
Int J Biol Macromol. 2023 Aug 1;245:125447. doi: 10.1016/j.ijbiomac.2023.125447. Epub 2023 Jun 16.
Carboxypeptidase A (CPA) with efficient hydrolysis ability has shown vital potential in food and biological fields. In addition, it is also the earliest discovered enzyme with Ochratoxin A (OTA) degradation activity. Thermostability plays an imperative role to catalyze the reactions at high temperatures in industry, but the poor thermostability of CPA restricts its industrial application. In order to improve the thermostability of CPA, flexible loops were predicted through molecular dynamics (MD) simulation. Based on the amino acid preferences at β-turns, three ΔΔG-based computational programs (Rosetta, FoldX and PoPMuSiC) were employed to screen three variants from plentiful candidates and MD simulations were then used to verify two potential variants with enhanced thermostability (R124K and S134P). Results showed that compared to the wild-type CPA, the variants S134P and R124K exhibited rise of 4.2 min and 7.4 min in half-life (t) at 45 °C, 3 °C and 4.1 °C in the half inactivation temperature (T), in addition to increase by 1.9 °C and 1.2 °C in the melting temperature (T), respectively. The mechanism responsible for the enhanced thermostability was elucidated through the comprehensive analysis of molecular structure. This study shows that the thermostability of CPA can be improved by the multiple computer-aided rational design based on amino acid preferences at β-turns, broadening its industrial applicability of OTA degradation and providing a valuable strategy for the protein engineering of mycotoxin degrading enzymes.
羧肽酶 A（CPA）具有高效的水解能力，在食品和生物领域具有重要的应用潜力。此外，它也是最早发现的具有赭曲霉毒素 A（OTA）降解活性的酶。热稳定性对于在工业中高温下催化反应至关重要，但 CPA 的热稳定性差限制了其工业应用。为了提高 CPA 的热稳定性，通过分子动力学（MD）模拟预测了柔性环。基于β-转角处氨基酸的偏好性，使用三个基于ΔΔG 的计算程序（Rosetta、FoldX 和 PoPMuSiC）从大量候选物中筛选出三个变体，然后使用 MD 模拟验证两个具有增强热稳定性的潜在变体（R124K 和 S134P）。结果表明，与野生型 CPA 相比，变体 S134P 和 R124K 在 45°C 时半衰期（t）分别提高了 4.2 分钟和 7.4 分钟，半失活温度（T）分别提高了 3°C 和 4.1°C，此外，熔点（T）分别提高了 1.9°C 和 1.2°C。通过对分子结构的综合分析，阐明了提高热稳定性的机制。本研究表明，基于β-转角处氨基酸的偏好性，通过多种计算机辅助的合理设计可以提高 CPA 的热稳定性，拓宽其 OTA 降解的工业应用，并为真菌毒素降解酶的蛋白质工程提供了有价值的策略。
