Liu Zihan, Liu Yiheng, Jiang Qixuan, Xu Haijun, Liu Luo
Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, PR China.
ChemistryOpen. 2025 Feb;14(2):e202400236. doi: 10.1002/open.202400236. Epub 2024 Oct 25.
L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.
L-天冬氨酸-α-脱羧酶（ADC）催化L-天冬氨酸脱羧生成β-丙氨酸，这是β-丙氨酸生物合成中的决定性步骤。然而，ADC较低的催化稳定性和效率限制了其工业应用。在本研究中，以枯草芽孢杆菌的一种ADC变体作为工程改造的起点。通过易错PCR构建随机诱变文库，随后进行高通量筛选，鉴定出四个取代位点（S7N、K63N、A99T和K113R）。通过对这些位点的饱和诱变文库进行筛选和计算分析，获得了两个性能得到改善的重组变体N3（S7N/K63N/I88M/A99E/K113R/I126*）和Y1（S7Y/K63N/I88M/A99E/K113R/I126*）。与野生型相比，最佳的两个变体的催化效率和催化稳定性分别提高了95%（变体N3）和89%（变体Y1）。此外，Y1的半衰期提高了3.37倍，总周转数提高了2倍。亲水性分析和分子动力学（MD）模拟表明，关键氨基酸残基亲水性和空间位阻的增加会影响催化活性和稳定性。变体催化性能的提高可归因于它们在活性口袋内与底物结合能力的增强以及基于机制的失活的减轻。
