基于理性设计策略提高呕吐毒素解毒酶DepB的催化活性

Enhancing the catalytic activity of deoxynivalenol-degrading enzyme DepB through rational engineering

[目的]呕吐毒素(DON)广泛存在于粮谷类原料及其制品中,对人和动物的健康都造成严重危害。然而,现有呕吐毒素解毒酶DepB存在种类少、催化性能差等问题,限制了其在食品加工和饲料中的应用。本研究采用理性设计策略对呕吐毒素解毒酶DepB进行分子改造,以提高DepB的催化活性。[方法]通过对底物10范围内的氨基酸残基进行丙氨酸和甘氨酸扫描,确定突变“热点”氨基酸残基。随后,采用定点饱和突变和组合突变技术筛选得到催化活性显著提高的突变酶,并对其进行三维结构模拟和分子动力学模拟,分析其催化活性提高的分子机制。[结果]通过筛选获得了催化活性显著提高的三点突变酶A289V/A227R/S54N,该酶比活力为335.77 U·mg^(-1),是野生型DepB的2.25倍。三点突变酶的K m值由95.11μmol·L^(-1)下降到58.33μmol·L^(-1),k cat/K m值由5.99 mmol^(-1)·L·s^(-1)提高至16.94 mmol^(-1)·L·s^(-1),亲和力和催化效率均显著提高。通过三维结构模拟和分子动力学模拟分析,发现空间位阻减小、静电相互作用增强、酶-底物-辅酶结合自由能降低是其催化活性提高的分子机制。[结论]基于理性设计策略提高了DepB的催化活性,强化了DepB的适用性能,为呕吐毒素解毒酶的分子改造及其在食品加工和饲料中的应用提供依据。

[Objectives]Deoxynivalenol(DON)frequently occurs in cereal grain crops and their products,posing a serious health safety threat to humans and livestock.However,existing deoxynivalenol-degrading enzymes suffer from scarce species and poor catalytic activity,which restrict its potential for industrial application.Herein,rational engineering with a structure-based design was used to improve the catalytic activity of deoxynivalenol-degrading enzyme DepB.[Methods]Amino acid residues within the range of 10 around the natural substrate for alanine and glycine scanning were selected.“Hot spot”amino acid residues were then screened and subjected to targeted saturation mutagenesis.The best variant enzyme was further obtained by recombining the improved mutations sites.3D-structure simulation and molecular dynamics simulation were carried out to explain reasons for the improvement of its catalytic efficiency.[Results]The three-point best variant enzyme(A289V/A227R/S54N)was finally screened and its specific activity was 335.77 U·mg^(-1),which showed a 2.25-fold increased specific activity.K m values of three-point variant enzyme decreased from 95.11μmol·L^(-1)to 58.33μmol·L^(-1),while k cat/K m values increased from 5.99 mmol^(-1)·L·s^(-1)to 16.94 mmol^(-1)·L·s^(-1).It was obvious that its affinity for the substrate and catalytic efficiency were both markedly improved when compared with the wild type.3D-structure simulation and molecular dynamics simulation were conducted to understand the mechanism of the activity change behind the best triple-sites mutant,and it was found that variations in steric hindrance,enhanced electrostatic interactions in the substrate binding pocket,and decreased enzyme-substrate-coenzyme binding energy were the chief factors contributing to the increased catalytic activity.[Conclusions]A rational design was utilized to significantly enhance the catalytic activity and applicability of DepB,thus forming a foundation for molecular modification of deoxynivalenol-degrading enzymes and potential industrial application.