Construction and application of expression system for high-throughput screening of β-glucuronidase with high bond selectivity

doi:10.11949/j.issn.0438-1157.20150788

Abstract

Abstract:

To improve the bond selectivity of β-glucuronidase towards glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG), a one-pot selecting system (CELS) was established for the high-throughput screening. The system is consisted of the constitutive expression of fungal β-glucuronidase and the bacteriophage lyase SRRz under the temperature-induced promoter in E. coli. Then, the expression efficiency of PGUS-E with promoters of different strengths as well as the effect of the expression of bacteriophage lyase SRRz on lysis were investigated. In addition, a directed evolution based on the established screening system (CELS) was introduced to improve the bond selectivity of PGUS-E in vitro. The results indicated that CELS effectively integrated the strain culture, protein expression, cell lysis and the reaction. And a series of positive mutants with improved bond selectivity towards GAMG were obtained. It is concluded that the changes in α helix structure of TIM barrel domain have a strong influence on the improvement of the bond selectivity.

Key words: β-glucuronidase, biocatalysis, molecular biology, optimal design, bond selectivity

摘要：

分子改造是提高β-葡萄糖醛酸苷酶(PGUS-E)在生产单葡萄糖醛酸基甘草次酸(GAMG)过程中的键选择性的有效方法,但高通量筛选方法的缺失是限制其发展的重要瓶颈。为解决该问题,在大肠杆菌中构建了组成型表达β-葡萄糖醛酸苷酶(PGUS-E),利用温度诱导表达噬菌体裂解酶SRRz实现原位裂解的一锅法(one-pot)甘草酸键选择性筛选体系(CELS);同时考察了启动子强度对PGUS-E外源表达的效果,优化了不同温度下裂解酶SRRz的表达对酶释放效率的影响。结果表明,CELS筛选体系成功实现了集培养、产酶、裂解和反应于一体化工艺,并将其应用于PGUS-E的非理性改造,获得了一系列键选择性显著提高的突变酶。研究证明,TIM 桶状结构域中α螺旋结构的变化对甘草酸键选择性的改善有很大的影响。

关键词: β-葡萄糖醛酸苷酶, 生物催化, 分子生物学, 优化设计, 键选择性

CLC Number:

Q816

Lü Bo, FENG Xudong, LI Chun. Construction and application of expression system for high-throughput screening of β-glucuronidase with high bond selectivity[J]. CIESC Journal, 2015, 66(8): 3189-3194.

吕波, 冯旭东, 李春. β-葡萄糖醛酸苷酶键选择性高效筛选体系的构建与应用[J]. 化工学报, 2015, 66(8): 3189-3194.

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