Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase.
- Author:
Hongli PU
;
Bo LÜ
;
Dongxu ZHAO
;
Chun LI
- Publication Type:Journal Article
- MeSH:
Chromatography, High Pressure Liquid;
Chromatography, Thin Layer;
Escherichia coli;
metabolism;
Glucuronidase;
chemistry;
Industrial Microbiology;
Mutagenesis;
Recombinant Proteins;
chemistry;
Temperature
- From:
Chinese Journal of Biotechnology
2015;31(7):1119-1128
- CountryChina
- Language:Chinese
-
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.
