联合突变对α-L-鼠李糖苷酶性质的影响

Effects of Combined Mutations on the Properties of α-L-Rhamnosidase

摘要:

目的 提升α-L-鼠李糖苷酶（EC 3.2.1.40）的催化活性、热稳定性和pH稳定性。方法 基于已改良的单点突变体，构建了3个联合突变体：K423R-R307Y-D508N、K423R-R307Y-N561D和K389R-R307Y-N561D。结果 相比于原始酶（wild type，WT），K423R-R307Y-D508N在温度为65、70 ℃时的半衰期均提高了0.24 min，在pH为 7.0、8.0时的半衰期分别提高了145.64、4.91 min，但其相对酶活力仅为WT的41.01%；K423R-R307Y-N561D在pH为7.0、8.0时的半衰期分别提高了51.42、20.65 min，相对酶活力为WT的63.26%；K389R-R307Y-N561D的相对酶活力是WT的167.20%，在温度为65、70 ℃时的半衰期分别提高了1.60、1.34 min，但在碱性环境下的pH稳定性未发生明显变化。通过分子对接、分子动力学模拟和MM-PBSA分析发现，K389R-R307Y-N561D的催化结构域Loop区和底部部分α螺旋结构的柔性增大，使酶与底物结合更充分，提升了亲和性；但R307突变引发局部扰动，破坏氢键网络及π-π疏水相互作用，导致结构稳定性下降。结论 联合突变触发位点补偿机制是α-L-鼠李糖苷酶催化活性与结构稳定性难以协同提升的主要原因。

Abstract:

Objective This study aims to enhance the catalytic activity, thermal stability, and pH stability of α-L-rhamnosidase (EC 3.2.1.40).Method Three combined mutants (K423R-R307Y-D508N, K423R-R307Y-N561D, and K389R-R307Y-N561D) were constructed based on improved single-point mutants.Result Compared with the wild type (WT), the combined mutants K423R-R307Y-D508N exhibited an increased half-life of 0.24 min at both 65 ℃ and 70 ℃. Additionally, its half-life improved by 145.64 min and 4.91 min at pH 7.0 and pH 8.0, respectively. However, its relative enzyme activity was only 41.01% of that of the WT. The combined mutant K423R-R307Y-N561D exhibited an increased half-life of 51.42 min and 20.65 min at pH 7.0 and pH 8.0, respectively, and its relative enzyme activity were 63.26% of that of the WT. The combined mutant K389R-R307Y-N561D achieved an relative enzyme activity 167.20% of that of the WT, with an increased half-life of 1.60 min and 1.34 min at 65 ℃ and 70 ℃, respectively. However, its stability in alkaline environments did not show significant improvement. Molecular docking, molecular dynamics simulations, and MM-PBSA analyses revealed that the increased flexibility of the catalytic domain Loop region and partial α helix structure at the bottom of K389R-R307Y-N561D enhanced substrate binding and affinity. However, the R307 mutation induced local perturbations, disrupting the hydrogen bond network and π-π hydrophobic interactions, leading to decreased structural stability.Conclusion The site compensation mechanism triggered by combined mutations is the primary reason for the difficulty in simultaneously enhancing the catalytic activity and structural stability of α-L-rhamnosidase.