Rational Design Engineering of 5-Aminolevulinate Synthase with Activity and Stability Enhancement

5-Aminolevulinic acid synthase (ALAS) is the key rate-limiting enzyme in the synthesis of the vital biosynthetic intermediate 5-aminolevulinic acid (ALA). However, its catalytic efficiency is compromised due to its low activity and poor stability. Here, we obtained the mutant I325M/V390Y/H391I (T6), which exhibited a 7.0-fold increase in specific activity (2.53 U/mg) compared to the wild type through the application of isothermal compressibility (βT) perturbation engineering in conjunction with two thermal stability prediction algorithms. Moreover, molecular dynamics simulations indicate that positive changes in intermolecular interactions, the substrate channel, and the binding pocket account for the improved catalytic activity of T6. Furthermore, T6 was immobilized on magnetic chitosan nanoparticles, maintaining 73.5% of its original activity after 10 reaction cycles. Overall, combination approaches were employed to construct a superior ALAS variant, providing a novel concept for the synthesis of ALA and a valuable benchmark for optimizing industrial enzymes in related fields.