title: [Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase].
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.
authors.forename: Hongli
authors.initials: H
authors.lastname: Pu
authors.forename: Bo
authors.initials: B
authors.lastname: Lü
authors.forename: Dongxu
authors.initials: D
authors.lastname: Zhao
authors.forename: Chun
authors.initials: C
authors.lastname: Li
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000378:metabolism; D005966:Glucuronidase / Q000737:chemistry*; D007218:Industrial Microbiology; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D005966:Glucuronidase