[Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase]

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.