基于多重计算设计策略提高奇异变形杆菌脂肪酶的热稳定性

Improving the thermal stability of Proteus mirabilis lipase based on multiple computational design strategies

摘要:

来自奇异变形杆菌的脂肪酶(Proteus mirabilis lipase,PML)具有较好的有机溶剂耐受性,在生产生物柴油等领域有广泛的应用潜力。然而,其热稳定性仍有待提高以更好地适应工业生产。近年来,基于计算机辅助的多种计算设计策略的发展为酶的热稳定性改造提供了更多的思路。文中首先选择了3种基于互补算法的软件ABACUS、PROSS以及FoldX为正筛对PML进行计算设计,选择其两两交集,利用PSSM序列保守性分析和G_REMLIN共进化分析两种负筛进一步缩小突变文库,得到18个单点突变设计。经实验验证,有7个T_m提高的突变体,其中K208G和G206D的ΔT_m最高,分别为3.75℃和3.21℃。选择其中5个酶活高于野生型的突变体利用贪婪累积策略进行组合,最终五点组合突变体M10的T_m提高了10.63℃,相对酶活为野生型的140%。组合过程中K208G和G206D展现出了一定的上位性,为M10热稳定性的提高作出了主要贡献。分子动力学模拟表明在突变位点及附近区域有新的作用力产生,G206D/K208G附近作用力的重排可能稳定了对PML稳定性有关键作用的Ca²⁺结合位点。本研究为天然酶的热稳定性改造提供了高效且兼顾用户友好性的计算设计方案,并为PML的改造及其工业应用的拓展奠定了基础。

Abstract:

Proteus mirabilis lipase (PML) features tolerance to organic solvents and great potential for biodiesel synthesis.However,the thermal stability of the enzyme needs to be improved before it can be used industrially.Various computational design strategies are emerging methods for the modification of enzyme thermal stability.In this paper,the complementary algorithm-based ABACUS,PROSS,and FoldX were employed for positive selection of PML mutations,and their pairwise intersections were further subjected to negative selection by PSSM and G_REMLIN to narrow the mutation library.Thereby,18 potential single-point mutants were screened out.According to experimental verification,7 mutants had melting temperature (T_m) improved,and the ΔT_m of K208G and G206D was the highest,which was 3.75℃ and 3.21℃,respectively.Five mutants with activity higher than the wild type (WT) were selected for combination by greedy accumulation.Finally,the T_m of the five-point combination mutant M10 increased by 10.63℃,and the relative activity was 140% that of the WT.K208G and G206D exhibited certain epistasis during the combination,which made a major contribution to the improvement of the thermal stability of M10.Molecular dynamics simulation indicated that new forces were generated at and around the mutation sites,and the rearrangement of forces near G206D/K208G might stabilize the Ca²⁺ binding site which played a key role in the stabilization of PML.This study provides an efficient and user-friendly computational design scheme for the thermal stability modification of natural enzymes and lays a foundation for the modification of PML and the expansion of its industrial applications.