β-葡萄糖醛酸苷酶键选择性高效筛选体系的构建与应用  

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

作　　者：吕波[1] 冯旭东[1] 李春[1] 

机构地区：[1]北京理工大学生命学院生物工程系,北京100081

出　　处：《化工学报》2015年第8期3189-3194,共6页CIESC Journal

基　　金：国家自然科学基金项目(21376028;21176028;21425624)~~

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。To improve the bond selectivity ofβ-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 fungalβ-glucuronidase and the bacteriophage lyase SRRz under the temperature-induced promoter inE. 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-Ein vitro. Theresults 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 inα helix structure of TIM barrel domain have a strong influence on the improvement of the bond selectivity.

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 

分 类 号：Q816[生物学—生物工程]