使用祖先序列重建 (ASR) 设计新型阿斯巴达蛋白酶 (NAP) 用于奶酪制造
Shilan S Saleem1,2,3, Oluwasola Michael Akinola4,2, Mohd Basyaruddin Abdul Rahman5
1Enzyme Technology and X-Ray Crystallography Laboratory, Institute of Bioscience, Universiti Putra Malaysia, VacBio 5, 43400, Serdang, Selangor, Malaysia.
Molecular biotechnology
|July 7, 2025
概括
计算酶工程设计了一种新型的阿斯巴达蛋白酶 (NAP),以改善奶酪生产. 与当前的牛奶凝固酶相比,这种工程酶表现出增强的特异性和降低的热稳定性,提供了更好的奶酪产量和质感的潜力.
科学领域:
- 酶学和蛋白质工程 酶学和蛋白质工程
- 计算生物学和生物信息学
- 食品科学与技术 食品科学与技术
背景情况:
- 树脂菌菌蛋白酶 (RMP) 是一种在奶酪生产中广泛使用的乳凝酶 (MCE).
- 目前的MCE如RMP具有局限性,包括非特异性蛋白质分解活性和高热稳定性,对奶酪质量和产量产生负面影响.
- 对于乳制品应用,需要改进的MCE,增强特异性和控制稳定性.
研究的目的:
- 通过计算设计一种具有改善 κ-素亲和力和降低热稳定性的新型亚斯巴拉特蛋白酶 (NAP).
- 通过酶工程解决现有的牛奶凝固酶的局限性.
- 开发下一代凝固剂用于工业乳制品应用,使用祖先序列重建 (ASR).
主要方法:
- 使用祖先序列重建 (ASR) 设计NAP的计算酶工程.
- 引入向氨基酸修饰酶的催化和片区域.
- 分子对接,分子动力学 (MD) 模拟和结构分析,以比较NAP与野生类型RMP.
- 结合性亲和度计算 (MM-GBSA) 和结构指标分析 (SASA,Rg,RMSD,RMSF,FEL,PCA). 这两种方法都非常有用.
主要成果:
- 与野生型RMP相比,设计的NAP表现出增强的基质相互作用和结合亲和力.
- NAP的热稳定性降低,灵活性增加,这表明在较温和的条件下性能有所改善.
- 在NAP-κ-casein复合体中形成了新的键和盐桥,表明相互作用动态发生了变化.
- 计算分析预测了NAP的提高特异性和减少非目标蛋白解.
结论:
- 通过ASR设计的新型酸蛋白酶 (NAP) 显示出作为优质乳凝剂的显著前景.
- NAP的增强特异性和受控的热稳定性为目前奶酪生产的局限性提供了潜在的解决方案.
- 计算酶设计,特别是ASR,是开发适合工业应用的定制酶的强大方法.
- 需要进一步的实验验证,以确认NAP在现实世界乳制品工艺中的有效性.
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