氨基基转移酶的工程揭示了静电相互作用在增强的催化活动中的关键作用
Weibin Lin1, Qiangqiang Wang1, Ruizhi Han2
1Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Applied biochemistry and biotechnology
|August 29, 2023
概括
研究人员设计了一种关键酶 - - 甲氨基基转移酶 (MAT),以改善S-甲氨基甲 (SAM) 的产生. 突变酶增强了酶活性和效率,克服了工业SAM合成的局限性.
科学领域:
- 生物化学和分子生物学
- 酶工程是什么? 酶工程是什么?
- 工业生物技术 工业生物技术
背景情况:
- S-adenosylmethionine (SAM) 是一种重要的食补充剂,由甲氨基基转移酶 (MAT) 合成.
- 由于产品抑制,MAT活动受到显著限制,阻碍了工业SAM生产.
- 由于较低的产品抑制,Bacteroides fragilis MAT (BfMAT) 被确定为一个潜在的候选者.
研究的目的:
- 通过蛋白质工程来增强BfMAT的催化活性和效率.
- 为了克服SAM合成中的产品抑制局限性.
- 研究静电相互作用在BfMAT的催化性能中的作用.
主要方法:
- 基因挖掘用于识别BfMAT.
- 分子对接以确定ATP附近的关键残留物.
- 局部导向的突变发生产生三重变异 (M3-1,M3-2,M3-3).
- 酶活性测定,动力分析 (Km,kcat/Km),以及最佳pH值的确定.
- 分子动力学 (MD) 模拟和相互作用分析.
主要成果:
- 三重变体显示显著更高的特定活动 (1.831.94 U/mg),比野生类型 (WT) 增加110.5125.6%.
- 变种M3-1和M3-3显示Km值降低 (31.460.6%的下降),并大大提高了催化效率 (kcat/Km增加了322.5681.1%).
- 所有变体的最佳pH从8.0转移到7.5,减少了与增强活性相关的静电相互作用.
结论:
- 工程BfMAT变体显示了显著改善的催化活性和效率.
- 减少突变部位的静电相互作用对于提高酶性能至关重要.
- 该战略为提高工业SAM生产中的MAT催化效率提供了一个有希望的方法.
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