Enhancement of thermostability and catalytic properties of ammonia lyase through disulfide bond construction and backbone cyclization

• Published in: International journal of biological macromolecules 2022-10, Vol.219, p.804-811
• Main Authors: Ni, Zi-Fu, Li, Na, Xu, Pei, Guo, Ze-Wang, Zong, Min-Hua, Lou, Wen-Yong
• Format: Article
• Language: English
• Subjects: Backbone cyclization; Disulfide bond; Methylaspartate ammonia-lyase; Thermostability
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2022.07.213

Ammonia lyases have great application potential in food and pharmaceuticals owing to their unique ammonia addition reaction and atom economy. A novel methylaspartate ammonia-lyase, EcMAL, from E. coli O157:H7 showed high catalytic activity. To further strengthen its thermostability and activity, disulfide bond and backbone cyclization (cyclase) variants were constructed by rational design, respectively. Among them, variant M3, with a disulfide bond introduced, exhibited a 2.3-fold increase in half-life at 50 °C, while cyclase variant M8 showed better performance, with 25.9-fold increases. The synergistic promotion effect of this combinational strategy on activity and stability was also investigated, and the combined mutant M9 exhibited a 1.1-fold improvement in catalytic efficiency while maintaining good thermostability. Circular dichroism analysis and molecular dynamics simulation confirmed that the main sources of improved thermostability were reduced atomic fluctuation and a more stable secondary structure. To our knowledge, this is the first example of combining the introduction of disulfide bonds with cyclase construction to improve enzyme stability, which was characterized by modification away from the enzyme active center, and provided a new method for adjusting enzyme thermostability. [Display omitted] •Methylaspartate ammonia lyase was thermostability enhancement for the first time.•Dual-strategy combination to improve the thermostability of enzyme.•Simultaneous improvements in catalytic efficiency and thermostability.•The thermostability improvement mechanism was analyzed using MD simulation and CD.