Institute for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, 100081, People's Republic of China.
Department of Microbiology, School of Life Sciences, Yan'an University, Yan'an, 716000, People's Republic of China.
Appl Microbiol Biotechnol. 2019 Jun;103(12):4813-4823. doi: 10.1007/s00253-019-09790-3. Epub 2019 May 4.
In this study, we aimed to shift the optimal pH of acidic β-glucuronidase from Aspergillus oryzae Li-3 (PGUS) to the neutral region by site-directed mutagenesis, thus allowing high efficient biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid (GA) under higher pH where the solubility of GL could be greatly enhanced. Based on PGUS structure analysis, five critical aspartic acid and glutamic acid residues were replaced with arginine on the surface to generate a variant 5Rs with optimal pH shifting from 4.5 to 6.5. The catalytic efficiency (k /K) value of 5Rs at pH 6.5 was 10.7-fold higher than that of PGUS wild-type at pH 6.5, even 1.4-fold higher than that of wild-type at pH 4.5. Molecular dynamics simulation was performed to explore the molecular mechanism for the shifted pH profile and enhanced pH stability of 5Rs.
在这项研究中，我们旨在通过定点突变将酸性β-葡萄糖醛酸酶（PGUS）的最适 pH 值从酸性区域转移到中性区域，从而在 pH 值更高的条件下实现甘草酸（GL）向甘草次酸（GA）的高效生物转化，因为在更高的 pH 值下 GL 的溶解度可以大大提高。基于 PGUS 结构分析，在表面上将五个关键的天冬氨酸和谷氨酸残基替换为精氨酸，从而产生了一种变体 5Rs，其最适 pH 值从 4.5 转移到了 6.5。在 pH 6.5 下，5Rs 的催化效率（k / K）值比野生型 PGUS 在 pH 6.5 下的高 10.7 倍，甚至比野生型在 pH 4.5 下的高 1.4 倍。进行了分子动力学模拟，以探索 5Rs 的 pH 值迁移和增强的 pH 值稳定性的分子机制。
