Variants of glycosyl hydrolase family 2 β-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with β-glucuronidase (β-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various β-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard β-glucuronidase substrates to activity on recalcitrant drug glucuronides.