Glucuronide synthesis is part of mammalian Phase II metabolism, in which detoxification of xenobiotics occurs. Glucuronides are therefore important as metabolite standards in various clinical tests. As such, the ability to produce glucuronides in the lab, in gram-scale, is of important relevance to many industries. A chemoenzymatic synthetic route was successfully developed via a point mutation E504G, a residue in the catalytic duet of β-glucuronidase (β-GUS), to produce the synthetic enzyme, glycosynthase (SynE504G). ⁽¹⁾ However, the reaction typically takes 2-3 days, while yields for bulkier and more hydrophobic substrates such as testosterone remain low. Addition of t-butanol (t-BuOH) has shown to benefit the reaction, in large part due to increased solubility of the substrates. However, both β-GUS and its derived SynE504G are only tolerant of t-BuOH up to 20%. Hence, it would have been beneficial to increase the solvent stability of the synthase. This was accomplished using a medley of directed evolution strategies involving site saturated mutagenesis (SSM) and targeted random mutagenesis of β-GUS. Several mutants that confer increased activity were identified and characterized.