单点突变重塑远端盐桥提升植酸酶的热稳定性

A single-point mutation enhances phytase thermostability through remodeling distant salt bridges

摘要:

植酸酶(phytase, EC 3.1.3.8)是一种可以特异性水解植酸的酶，其产物为正磷酸盐、肌醇磷酸异构体及游离肌醇，是一种具有前景的食品用酶。本研究为了提高来源于塔宾曲霉(Aspergillus tubingensis)的植酸酶的热稳定性，通过FirePort平台预测突变对蛋白稳定性的影响，结合FoldX和Rosetta工具的突变自由能进行突变筛选，最终构建了4种不同的突变体，并筛选出最优的突变体T273K表征其酶学性质。突变体T273K在60 ℃的半衰期比野生型提高了67.56%，80 ℃时的半衰期相对于野生型提高了92.86%，催化效率(k_cat/K_m)为196.11 L/(μmol·min)，相对野生型提高了50.56%。这些结果均说明突变体的热稳定性及活力均有所提高。动力学模拟分析发现，突变体T273K远端片段(205-210，380-400)的盐桥数量显著增加，导致蛋白质整体刚性增加，进而提升整体蛋白质热稳定性。本研究成功筛选出一个突变体T273K，有效提高了植酸酶的热稳定性，并通过动力学模拟分析发现局部突变体对远端片段盐桥进行重塑，从而提升了蛋白质整体刚性使得热稳定性增加，为后续有关酶热稳定性改造提供了一种有效的路径。

Abstract:

Phytase (EC 3.1.3.8) is an enzyme that specifically hydrolyzes phytic acid, yielding products such as orthophosphate, inositol phosphate isomers, and free inositol. It is a promising enzyme for food applications. This study aims to enhance the thermostability of the phytase derived from Aspergillus tubingensis. The FireProt platform was employed to predict the impacts of mutation sites on protein stability, which was complemented by FoldX and Rosetta-based calculations of mutation free energy changes for mutant screening. Four distinct mutants were subsequently constructed. The elite mutant T273K was screened out for enzymatic characterization. The half-life of T273K was increased by 67.56% at 60 ℃ compared with that of the wild type (WT), and by 92.86% at 80 ℃. The catalytic efficiency (k_cat/K_m) of T273K was 196.11 L/(μmol·min), representing a 50.56% increase relative to that of WT. These results collectively demonstrated improved thermostability and activity of T273K. Kinetic simulation analysis revealed a significant increase in the number of salt bridges in the distal fragments (205-210 and 380-400) of T273K, which led to improved overall protein rigidity, thereby enhancing the thermostability. This study successfully identified a mutant T273K, which effectively enhanced the phytase thermostability. Furthermore, kinetic simulations revealed that a local mutation remodeled salt bridges in distal fragments to increase overall protein rigidity and improve the protein thermostability. This provides an effective strategy for future modifications aimed at enhancing enzyme thermostability.