E. coli BL21(DE3) expressing mutated Bl BSH at key active site residues exhibited altered deconjugation and conjugation activities. Deconjugation, indicated by the CA:TCA ratio, was assessed in M9 media with 1 mM TCA. Conjugation, measured through Ala-CA production, was evaluated in Luria–Bertani media with 1 mM TCA using LC–MS/MS. Controls included untransformed E. coli (Unt) and E. coli expressing unmutated Bl BSH (WT). Bars represent mean values ± s.d. (n = 3 biologically independent bacterial cultures).
