Immobilization of Staphylococcus aureus Sortase A on Chitosan Particles and Its Applications in Peptide-to-Peptide Ligation and Peptide Cyclization.

Chitosan macro-particles prepared by the neutralization method were applied to Sortase A (SrtA) immobilization using glutaraldehyde as a crosslinking agent. The particles were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Response surface methodology (RSM) was employed to optimize the immobilization process. An average specific activity of 3142 U (mg protein) was obtained under optimized immobilization conditions (chitosan concentration 3%, SrtA concentration 0.5 mg·mL, glutaraldehyde concentration 0.5%, crosslinking and immobilization at 20 °C, crosslinking for 3 h, and an immobilization time of 8 h). The transpeptidase activity of immobilized SrtA was proved by a peptide-to-peptide ligation with a conversion yield approximately at 80%, and the immobilized catalyst was successfully reused for five cycles without obvious activity loss. Moreover, the scale-up capability of using immobilized SrtA to catalyze a head-to-tail peptide cyclization was investigated in a batch reaction and the conversion yield was more than 95% when using 20 mg of peptide as a substrate.