计算机辅助设计以提高 ω-胺转氨酶对甲醇作为辅溶剂的耐受性
Shuai Qiu1, Tong Wang1, Cong-Lin Ju1
1Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China.
Bioorganic chemistry
|August 29, 2025
概括
在甲醇中改造的Aspergillus terreus ω-胺转氨酶 (AtATA) 的活性和稳定性得到了提高. 这种改进的酶对于工业生物技术中高效的 (R) - 基拉胺生产至关重要.
科学领域:
- 生物催化
- 酶工程
- 工业生物技术
背景情况:
- 有机溶剂中的酶稳定性是工业生物技术的一个主要障碍.
- 有机溶剂对酶催化有好处,但可以使酶失活.
- 甲醇耐受性 ω-氨基转氨酶是 (R) - 奇拉氨基的生产所需的.
研究的目的:
- 增强阿斯珀吉勒斯土壤 ω-胺转氨酶 (AtATA) 的活性和甲醇耐受性,用于 (R) - 基拉胺合成.
- 通过计算方法和定向进化来识别赋予改善酶特性的关键突变.
- 在扩大反应中验证工程酶的性能.
主要方法:
- 用计算机辅助的定向进化来识别潜在的突变部位.
- 组合变种M4 (AtATA-T23I/E117V/T200G/Q294K) 被生成并进行了表征.
- 进行了分子动力学模拟以了解增强甲醇耐药性的机制.
- 评估了酶活性,30%甲醇的半衰期和大规模转化.
主要成果:
- 与野生型AtATA相比,在30%的甲醇中,工程M4变体的催化活性高出19. 0倍,半衰期长出3. 5倍.
- 分子动力学模拟显示基质结合口袋中甲醇占用率降低和疏水性增加是改善耐受性的关键因素.
- 在50毫升尺度的实验中,基质转化从29.1%增加到76.7%,而 (R) - - - - 甲基乙烯的度超过99%.
结论:
- 改造的AtATA变种M4显示出显著改善的活性和甲醇耐受性.
- 计算和实验方法成功发现了在有机溶剂中增强酶性能的突变.
- 这种工程酶对有效和可扩展的 (R) - 奇拉胺的生产具有很大的前景.
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