The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Department of Biological and Agricultural Engineering, University of California, Davis 1 Shields Ave, Davis, CA 95616, USA.
Enzyme Microb Technol. 2024 Oct;180:110505. doi: 10.1016/j.enzmictec.2024.110505. Epub 2024 Aug 24.
Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus (ppTA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type ppTA (WT). Furthermore, the mutant enzyme ppTA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.
转氨酶（EC 2.6.1.X，TAs）是手性胺合成中的重要生物催化剂，在手性胺或非天然氨基酸的合成领域具有重要价值。然而，TAs 在合成手性胺或非天然氨基酸时存在酶活低、催化效率差的问题，这阻碍了其工业应用。在本研究中，采用半理性设计策略，研究了我们之前研究中发现的来自泛酸不动杆菌（ppTA）的新型 TA，以提高其对 2-酮丁酸的酶活。通过同源建模和分子对接，选择底物结合 S 口袋的四个周围位点作为潜在的突变位点。通过丙氨酸扫描和饱和诱变，最终获得了酶活显著提高的最佳突变体 V153A，比野生型 ppTA（WT）高 578%。此外，与 WT 相比，突变酶 ppTA-V153A 的温度和 pH 稳定性也略有提高。随后，该突变体用于转化 2-酮丁酸制备 L-2-氨基丁酸（L-ABA）。突变体可以耐受 300 mM 的 2-酮丁酸，转化率为 74%，为手性胺的制备奠定了坚实的基础。
