Development of the Esterase PestE for Amide Bond Synthesis Under Aqueous Conditions: Enzyme Cascades for Converting Waste PET into Tamibarotene

A growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97 % in 6 h for formation of N-benzylfuranamide as the model reaction. We demonstrate PestE's potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L F289A and F33L. F33L F289A increased conversion of N-benzylfuranamide by 1.2-fold, and F33L gave a 4-fold increase in conversion to tamibarotene.