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.
Keywords: Backbone cyclization; Disulfide bond; Methylaspartate ammonia-lyase; Thermostability.
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