TY - JOUR
T1 - Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase
AU - Pu, Hongli
AU - Lü, Bo
AU - Zhao, Dongxu
AU - Li, Chun
N1 - Publisher Copyright:
© 2015 by the Institute of Microbiology, the Chinese Academy of Sciences and the Chinese Society.
PY - 2015/7/25
Y1 - 2015/7/25
N2 - 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.
AB - 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.
KW - Bond selectivity
KW - Glycyrrhetinic acid monoglucuronide
KW - Site-saturation mutagenesis
KW - β-glucuronidase
UR - https://www.scopus.com/pages/publications/84940836608
U2 - 10.13345/j.cjb.140595
DO - 10.13345/j.cjb.140595
M3 - Article
C2 - 26647587
AN - SCOPUS:84940836608
SN - 1000-3061
VL - 31
SP - 1119
EP - 1128
JO - Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology
JF - Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology
IS - 7
ER -