基于高压模拟理性改造空腔提高RML脂肪酶热稳定性
首发时间:2020-01-23
摘要:米黑根毛霉脂肪酶(Rhizomucor miehei lipase)在工业领域具有重要的研究价值,利用蛋白质工程技术提高其热稳定性以拓宽应用范围是许多学者关注重点之一。本文在6个压力梯度 (1Bar, 100Bar, 500Bar, 1000Bar, 2000Bar, 4000Bar) 下分别进行分子动力学模拟,并从模拟轨迹中选取4个具有代表性的构象用以空腔识别与计算。选择构成空腔的合适氨基酸进行虚拟饱和突变,通过FoldX suite 5.0和Rosetta 3.10进行自由能计算,筛选了5个潜在有效突变体:Y20F,T21V,S24A,T149I和T198V。基于分子动力学模拟技术,从原子水平分析突变对结构的影响,并通过基因工程技术,引入突变,诱导表达后进行实验验证。结果显示5个突变体中,T21V和T149I稳定性均提高50%,T198V稳定性提高40%,S24A与Y20F稳定性均降低。本研究揭示了以高压为研究方法来选择构成空腔的目标氨基酸,并以自由能为筛选准则来选择合适的突变体,为提高蛋白质的稳定性提供了一种可行的策略。
关键词: 空腔 高压;分子动力学模拟; 理性设计 稳定性
For information in English, please click here
Remodeling Cavity Based on High-Pressure Molecular Dynamic Simulation to Improve Thermal Stability of RML Lipase
Abstract:Rhizomocor miehei lipase has important research value in the industrial field. It is one of the focuses of many scholars to use protein engineering technology to improve its thermal stability to broaden its application range. In this paper, molecular dynamics simulations are performed under 6 pressure gradients (1Bar, 100Bar, 500Bar, 1000Bar, 2000Bar, 4000Bar), and 4 representative conformations were selected from the simulation trajectory for cavity recognition and calculation. The appropriate amino acids constituting the cavity were selected for virtual saturation mutations, and free energy calculations were performed using FoldX suite 5.0 and Rosetta 3.10. Five potential mutants were screened: Y20F, T21V, S24A, T149I, and T198V. Based on molecular dynamics simulation technology, the effect of mutations on the structure is analyzed at the atomic level, and mutations are introduced through genetic engineering technology, and the expression is induced for experimental verification. The results showed that the stability of T21V and T149I increased by 50%, the stability of T198V increased by 40%, and the stability of S24A and Y20F decreased in the five mutants. This study revealed that high pressure was used as the research method to select the target amino acids that constituted the cavity, and free energy was used as the selection criterion to select the appropriate mutant, which provided a feasible strategy for improving the stability of the protein.
Keywords: Cavity High pressure Molecular dynamics simulation Rational design Stability
引用
No.****
动态公开评议
共计0人参与
勘误表
基于高压模拟理性改造空腔提高RML脂肪酶热稳定性
评论
全部评论0/1000