重组β-葡萄糖醛酸苷酶键选择性的半理性改造

Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase

摘要:

为了提高Escherichia coli重组表达的β-葡萄糖醛酸苷酶 (PGUS-E) 的键选择性，本研究以PGUS-E结构与功能关系的推测为指导，选择了可能影响PGUS-E的键选择性的R329、T369、N467位点进行定点饱和突变，利用薄层层析 (TLC) 和高效液相色谱 (HPLC) 对键选择进行筛选，得到优势突变酶R329K、T369V。结果显示：与PGUS-E酶相比，突变酶R329K、T369V键选择性分别提高26.9%、34.3%。突变酶的酶学性质研究表明，突变酶的最适pH和温度与PGUS-E一致，但其酶催化效率下降。由此可见，R329、T369对酶催化的键选择性和酶的活性有显著影响。综上结果，本文应用饱和突变方法改善了PGUS-E 的键选择性，为酶的结构和功能关系理解提供了实验依据。

Abstract:

To improve bond selectivity of recombinant β-glucuronidase in Escherichia coli (PGUS-E), based on the PGUS-E structure guidance, three key points R329, T369 and N467 were identified to be responsible for the bond selectivity of PGUS-E, and further saturation mutagenesis was conducted. Two positive mutants R329K and T369V were obtained by a combined selection technique of thin-layer chromatography and high performance liquid chromatography. Compared to PGUS-E, the bond selectivity of mutants R329K and T369V increased by 26.9% and 34.3%, respectively; whereas the biochemical properties such as pH and temperature profile were unchanged. Nevertheless, the activity was decreased compared to PGUS-E. These results further confirmed that sites R329 and T369 played important roles for the bond selectivity and activity. In summary, this study significantly increased the bond selectivity of PGUS-E by structure guided saturation mutagenesis, providing experimental support for elucidating the relationship between the structure and function of PGUS-E.