基于热不稳定区段分析和二硫键预测的玉米赤霉烯酮脱毒酶ZLHY-6热稳定性改造

Thermal stability modification of zearalenone detoxification enzyme ZLHY-6 based on thermolabile segment analysis and disulfide bond prediction

中文关键词:  玉米赤霉烯酮  脱毒酶ZLHY-6  热稳定性  定点突变  分子动力学模拟  热不稳定区段  二硫键

英文关键词:zearalenone  detoxification enzyme ZLHY-6  thermal stability  site-directed mutation  molecular dynamics simulations  thermolabile segment  disulfide bonds

中文摘要:

      目的 基于热不稳定区段分析和二硫键预测, 探索定点突变改造提高ZLHY-6的热稳定性的可行性。方法 基于分子动力学模拟设计两个策略以提高ZLHY-6的热稳定性。策略一: 寻找ZLHY-6热不稳定区段, 对区段内氨基酸进行计算机虚拟饱和突变, 根据突变自由能变(ΔG)筛选结果选取了10个突变体。策略二: 通过预测, 在ZLHY-6分子内引入二硫键, 每个突变体引入1对, 共计5个突变体。结果 突变体H134W和Q45C/A253C在45℃热处理20 min后, 其相对酶活分别为野生型的10倍和3.1倍, 并保留了对ZEN一定的活性(41.33%、102.67%)。通过分子动力学分析和结构模拟发现, 突变的引入可能削弱了构象的波动性, 提升了结构刚性, 从而提升了蛋白的热稳定性。结论 热不稳定氨基酸突变和二硫键构建在脱毒酶ZLHY-6改造上具有可行性, ZLHY-6热稳定性的增强提高了其在工业上的应用潜力, 这一结果也为探索酶的计算机辅助手段改造提供策略基础。

英文摘要:

      Objective To explore the feasibility of improving the thermal stability of ZLHY-6 by site-directed mutation based on thermolabile segment analysis and disulfide bond prediction. Methods Two strategies were designed to improve thermal stability of ZLHY-6 based on molecular dynamics simulations. Strategy 1: Find the thermally unstable segment of ZLHY-6, perform computer virtual saturation mutation on the amino acids in the segment, and select 10 mutants according to the screening results of mutation free energy change (ΔG). Strategy 2: By prediction, disulfide bonds were introduced into ZLHY-6 molecules, and one pair was introduced for each mutant, for a total of 5 mutants. Results The relative enzymatic activities of mutants H134W and Q45C/A253C were 10 times and 3.1 times that of the wild type, respectively, after heat treatment at 45℃ for 20 minutes, and they retained a certain amount of ZEN activity (41.33%, 102.67%). Through molecular dynamics analysis and structural simulations, it was found that the introduction of mutations may weaken the conformational volatility and enhance the structural rigidity, thereby enhancing the thermal stability of the protein. Conclusion Thermolabile amino acid mutation and disulfide bond construction are feasible in the transformation of detoxification enzyme ZLHY-6, the enhanced thermal stability of ZLHY-6 enhances its potential for industrial applications, and this result also provides a strategic basis for exploring computer-aided transformation of enzymes.