通过界面修改工程增强托酸氧甲基转移酶的热稳定性和酶活性
Xue Cai1,2, Xue Shi1,2, Jia-Ying Wang1,2
1The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, P. R. China.
Journal of agricultural and food chemistry
|May 30, 2024
概括
接口工程显著提高了酸酸甲基转移酶 (KPHMT) 的热稳定性,酸酸生产中的关键酶. 这一突破为工业应用提供了更好的蛋白质稳定性.
科学领域:
- 生物化学和分子生物学
- 蛋白质工程是指蛋白质工程.
- 工业生物技术 工业生物技术
背景情况:
- 松酸氧甲基转移酶 (KPHMT) 对于d-潘托酸生物合成至关重要.
- 大多数KPHMT的热稳定性较低,阻碍了蛋白质工程和工业生产.
- 之前的研究发现了来自*Corynebacterium glutamicum* (M0) 的高活性KPHMT突变体 (K25A/E189S).
研究的目的:
- 通过接口工程来提高KPHMT的热稳定性.
- 为了确定特定的突变,提高KPHMT的稳定性,用于工业应用.
- 了解改善热稳定的结构基础.
主要方法:
- 接口工程以无折叠能量计算,B因子分析和保存站点分析为指导.
- 对单点和双点接口突变物进行选.
- 热稳定性测试 (T1/2和Tm测量).
- 结构和分子动力学模拟.
主要成果:
- 五种接口突变 (E106S,E98T,E98N,S247I,S247D) 显示了改善的热稳定性.
- 双部位突变M8 (M0-E98N/S247D) 的T1/2增加了3.29倍,Tm从53.2°C增加到59.6°C.
- 模拟表明,由于表面静电变化和子单元间键增加,稳定性得到提高.
结论:
- 接口工程是改善KPHMT热稳定的有效策略.
- 这种M8突变体在提高工业d-pantothenic acid合成方面具有显著的潜力.
- 这项研究为KPHMT和其他酶的进一步蛋白质工程工作提供了基础.
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