Esterase Panel Development for Biocatalytic Amidation
Amide bonds are a key chemical functional group in a host of synthetic molecules, including pharmaceuticals. Amide bond formation is a resource-intensive transformation using traditional chemical synthesis. This generally requires ‘activation’ of a carboxylic acid and these reactions are performed with super-or stoichiometric amounts of expensive coupling reagents using atom-inefficient synthetic routes. The reagents, as well as the resultant waste, are highly toxic and environmentally unfriendly. On the other hand, the formation of amide bonds using enzymes is highly efficient and performed under mild conditions without the need for coupling agents. Proteases and lipases are known for amide formation under mild conditions. However, these enzymes are not promiscuous to accept a wide variety of substrates and also known to have a poor expression in prokaryotic systems. Here we report the identification of a microbial esterase (T. fusca, EST-031) and a panel of evolved enzyme variants of T. fuscaesterase that have a good prokaryotic expression with the ability to form amide bonds on a diverse set of ester and amine substrates including less reactive hindered amines. The evolved esterases were found to have low E. coli toxicity, good growth and high protein expression levels. These enzymes were found to have activity in not only aqueous systems but also in the presence of organic co-solvent.