合理工程的同精氨酸合成酶,以提高对精氨酸合成的催化效率
Wenjing Liu1, Xiaoxiang Hu1, Yi Yan1
1The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
Synthetic and systems biotechnology
|May 3, 2024
概括
设计了一种单一的酶 - - 荷马精氨酸合成酶,从前体中有效地产生精氨酸. 这种新的方法克服了目前用于精子胺生物合成的多酶过程的局限性.
科学领域:
- 生物催化剂是一种生物催化剂.
- 酵素工程是什么? 酶工程是什么
- 代谢工程是代谢工程.
背景情况:
- 精子胺是一种具有治疗潜力的重要聚胺.
- 现有的精子胺生物合成方法是低效和复杂的.
- 同类精氨酸合成酶 (HSS) 是精氨酸生产中的一个关键酶.
研究的目的:
- 来自Blastochloris viridis* (BvHSS) 的同类精子胺合成酶 (HSS) 进行工程,以实现高效的精子胺合成.
- 为了确定增强BvHSS活动和产品产量的特定突变.
- 阐明改善催化功能的结构基础.
主要方法:
- 基于序列结构功能分析的*Bv*HSS的局部定向突变发生.
- 酶活性测定用于确定特定活动,最佳pH值和温度.
- 分子对接和动力学模拟以分析基质结合和通道相互作用.
- 全细胞催化实验用于精子胺的生产.
主要成果:
- 与野生类型 (8.72 U/mg) 相比,工程 *Bv*HSS 突变 (D361E 和 E232D-D) 的特异性活动显著增加 (46.04 和 48.30 U/mg).
- 突变者在pH9.0和50°C时表现出最佳活动.
- 结构模拟显示,D361E突变缩小了结合口袋,而E232D突变扩大了基质通道.
- 全细胞催化在6小时内实现了725.3 mg/L (D361E) 和933.5 mg/L (E232D-D) 的精氨酸产量.
结论:
- 开发了一种实用的单酶方法来合成精子胺.
- 确定了关键的氨基酸残留物 (D361和E232),对*Bv*HSS的催化效率和基质通道至关重要.
- 提供了对优化聚胺生产的酶工程策略的见解.
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