分子工程 L-天门冬氨酸-α-脱羧酶,提高催化稳定性和性能。

IF 2.5 4区 化学 Q2 CHEMISTRY, MULTIDISCIPLINARY ChemistryOpen Pub Date : 2024-10-25 DOI:10.1002/open.202400236
Zihan Liu, Yiheng Liu, Qixuan Jiang, Haijun Xu, Luo Liu
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引用次数: 0

摘要

L-天门冬氨酸-α-脱羧酶(ADC)催化 L-天门冬氨酸脱羧生成 β-丙氨酸,这是生物合成 β-丙氨酸的决定性步骤。然而,ADC 的催化稳定性和效率较低,限制了其工业应用。本研究以枯草芽孢杆菌(Bacillus subtilis)的 ADC 变体为工程起点。通过易错 PCR 构建随机诱变文库,然后进行高通量筛选,最终确定了四个取代位点(S7 N、K63 N、A99T 和 K113R)。通过筛选这些位置上的饱和诱变文库和计算分析,得到了两个性能更好的重组变体 N3(S7 N/K63 N/I88 M/A99E/K113R/I126*)和 Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*)。与野生型相比,最好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,总周转次数提高了 2 倍。亲水性分析和分子动力学(MD)模拟显示,关键氨基酸残基亲水性和立体阻碍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的结合能力增强,以及基于机制的失活现象得到缓解。
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Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

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.

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来源期刊
ChemistryOpen
ChemistryOpen CHEMISTRY, MULTIDISCIPLINARY-
CiteScore
4.80
自引率
4.30%
发文量
143
审稿时长
1 months
期刊介绍: ChemistryOpen is a multidisciplinary, gold-road open-access, international forum for the publication of outstanding Reviews, Full Papers, and Communications from all areas of chemistry and related fields. It is co-owned by 16 continental European Chemical Societies, who have banded together in the alliance called ChemPubSoc Europe for the purpose of publishing high-quality journals in the field of chemistry and its border disciplines. As some of the governments of the countries represented in ChemPubSoc Europe have strongly recommended that the research conducted with their funding is freely accessible for all readers (Open Access), ChemPubSoc Europe was concerned that no journal for which the ethical standards were monitored by a chemical society was available for such papers. ChemistryOpen fills this gap.
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