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

We describe a novel, organic cosolvent-stable and cation-independent engineered enzyme for peptide coupling reactions. The enzyme is a variant of a stable calcium-independent mutant of subtilisin BPN, with the catalytic Ser212 mutated to Cys and Pro216 converted to Ala. 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