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

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.