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Updated: Feb 21, 2026

Modeling an Enzyme Active Site using Molecular Visualization Freeware
Published on: December 25, 2021
Design and characterization of new β-glucuronidase active site variants with altered substrate specificity
Ye Li1, Zhiqing Wang1, Patrick C Cirino2
1Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, 77204, USA.
Objective:
To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.
Results:
Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-β-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant.
Conclusions:
The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.
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