Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

There is a large and growing interest in pharmaceutical peptides over the past decades, with about 60 approved peptide drugs currently on the market and some 140 candidates in different phases of clinical development. By 2018, the worldwide peptide drugs market is expected to reach $18 billion. The importance of peptides is also recognized in the nutritional and cosmetic industries. Despite the high demand for peptides, their large-scale manufacturing remains challenging. Chemoenzymatic peptide synthesis (CEPS), wherein chemically synthesized peptide segments are ligated enzymatically, is potentially one of the most costefficient technologies for the synthesis of mediumsized and long peptides (20–50 amino acids). As compared to chemical segment coupling, CEPS offers several notable advantages, i.e., protection of the side-chain functionalities is not necessary, racemization is absent, and it is more environmentally friendly. Nevertheless, CEPS is currently rarely applied in academia and industry since it is not broadly applicable and suffers from severe hydrolytic side-reactions.
The enzyme, called peptiligase, catalyzes exceptionally efficient peptide coupling in water with a surprisingly high synthesis over hydrolysis (S/H) ratio. The S/H ratio of the peptide ligation reaction is correlated to the length of the peptide substrate and proved to be >100 for the synthesis of a 13-mer peptide, which corresponds to >99% conversion to the ligated peptide product and <1% hydrolytic side-reaction. Furthermore, peptiligase does not require a particular recognition motif resulting in a broadly applicable and traceless peptide ligation technology. Peptiligase is very robust, easy to produce in Bacillus subtilis, and its purification is straightforward. It shows good activity and stability in the presence of organic cosolvents and chelating or denaturing agents, enabling the ligation of poorly soluble (hydrophobic) or folded peptides. This enzyme could be useful for the (industrial) synthesis of diverse (pharmaceutical) peptides. In addition, peptiligase is able to efficiently catalyze head-to-tail peptide cyclization reactions.