Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

Protein biomaterials would have the potential to address global challenges in health and environment. Numerous production methods of the biomaterials exist, with sortase-mediated ligation (SML) being one of the representative technique. SML facilitates the site-specific conjugation of two compounds: donor peptides or proteins with a cell wall sorting signal at their C-terminus and nucleophiles that have oligoglycine at their N-terminal. In our research, we reconstructed an ancestral sortase E, named AcSE5, through a combination of sequence data mining and ancestral sequence reconstruction. AcSE5, a Ca2+ independent sortase, recognizes donors with LAETG at their C-termini and can employ both peptides bearing GGG- or GAA-at N-terminus and straight-chained alkylamines as nucleophiles. The enzyme can achieve efficient peptide conjugation, exceeding 70% under optimal conditions. With AcSE5, we synthesized two protein conjugates: Venus-conjugated shark variable new antigen receptor (VNAR) and dual-conjugated VNAR via poly(ethylene) glycol diamine. Direct enzyme immobilization to amino-terminated polystyrene beads was also achieved using AcSE5. The resultant beads, when conjugated with hyper-thermostable ancestral l-amino acid oxidases (HTAncLAAO2), can be employed for deracemization of various racemic amino acids into d-form. For three of phenylalanine derivatives, preparative-scale (100 mg scale) deracemization can be achieved. This process provides high enantiopurity (>99% ee) and isolation yields (>64%) through chemoenzymatic reactions. The immobilized HTAncLAAO2 showed complete resistance to 10 repeated reactions for a total of 240 h. AcSE5 is an excellent enzyme for SML applications.