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

Abstract

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 Ca 2+ independent sortase, recognizes donors with LAETG at their Ctermini 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.