Du Shuang, Zheng Nan, Zhang Zehua, Zhang Chenhao, Zhou Huimin, Deng Yu, Yin Jian, Cai Yongchao, Xia Xiaole
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
J Agric Food Chem. 2025 Jan 22;73(3):1892-1901. doi: 10.1021/acs.jafc.4c07294. Epub 2025 Jan 7.
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 (β) 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.
5-氨基乙酰丙酸合酶（ALAS）是重要生物合成中间体5-氨基乙酰丙酸（ALA）合成过程中的关键限速酶。然而，由于其活性低和稳定性差，其催化效率受到影响。在此，我们通过应用等温压缩性（β）扰动工程结合两种热稳定性预测算法，获得了突变体I325M/V390Y/H391I（T6），与野生型相比，其比活性提高了7.0倍（2.53 U/mg）。此外，分子动力学模拟表明，分子间相互作用、底物通道和结合口袋的正向变化是T6催化活性提高的原因。此外，T6被固定在磁性壳聚糖纳米颗粒上，经过10个反应循环后仍保持其原始活性的73.5%。总体而言，采用组合方法构建了一种优良的ALAS变体，为ALA的合成提供了新的概念，并为相关领域工业酶的优化提供了有价值的基准。
