重组β-葡萄糖醛酸苷酶键选择性的半理性改造
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国家科学自然基金 (Nos. 20976014, 21376028),国家杰出青年科学基金 (No. 21425624),高等学校博士学科点专项科研基金 (No. 20121101110050) 资助。


Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase
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National Natural Science Foundation of China (Nos. 21176028, 21376028); National Science Fund for Distinguished Young Scholars of China (No. 21425624), Doctoral Fund of Ministry of Education of China (No. 20121101110050).

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    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.

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普鸿丽,吕波,赵东旭,李春. 重组β-葡萄糖醛酸苷酶键选择性的半理性改造[J]. 生物工程学报, 2015, 31(7): 1119-1128

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  • 收稿日期:2014-12-02
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  • 在线发布日期: 2015-07-10
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