中文摘要：

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p H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。

英文摘要：

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 R329 K and T369 V were obtained by a combined selection technique of thin-layer chromatography and high performance liquid chromatographyCompared to PGUS-E, the bond selectivity of mutants R329 K and T369 V increased by 26.9% and 34.3%, respectivelywhereas the biochemical properties such as p H and temperature profile were unchanged. Nevertheless, the activity wadecreased compared to PGUS-E. These results further confirmed that sites R329 and T369 played important roles for thbond selectivity and activity. In summary, this study significantly increased the bond selectivity of PGUS-E by structurguided saturation mutagenesis, providing experimental support for elucidating the relationship between the structure anfunction of PGUS-E.