Scheme 2 Figure In our work on ACLs, we observed that these enzymes generally exhibit stability and tolerance across a wide range of buffers and pH levels, with optimal activity around pH 8. Both Mn and Mg cations were added to ensure the optimum activity of the enzymes under the screening reaction conditions, as we have previously observed that ACLs can have a preference for one or the other of the cations. From the 12 mono and dicarboxylic acids, 11 were found to be substrates of at least one ACL, although only traces of the corresponding amide were formed when dicarboxylic malonic (10b) and succinic acids (11b) were used (Figure ). None of the selected enzymes was active towards fumaric acid (12b). As expected, AMFC proved to be as sufficiently good nucleophile to allow amide formation via noncatalyzed addition to the adenylate intermediate. All the selected ACLs demonstrated activity towards most of the substrates. Among the eight enzymes, four exhibited distinct profiles: as already observed, MsACL demonstrated a preference for short aliphatic carboxylic acids, while ChACL showed activity mostly towards long aliphatic carboxylic acids. GtACL displayed a broad substrate range, being active towards all the tested monocarboxylic acids, although it yielded low result with aromatic substrate 7b. The most promiscuous enzyme, TsACL, facilitated the conversion of all three types of substrates: short-to long-chain monocarboxylic acids, monocarboxylic acids with a phenyl moiety, and succinate, a dicarboxylic acid. Analytical yields were determined by coupled enzymatic assay (Scheme S1), giving moderate to excellent results (Figure , Table ).