Abstract
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)−1 was obtained under optimized immobilization conditions (chitosan concentration 3%, SrtA concentration 0.5 mg·mL−1, 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.
Highlights
Sortase A (SrtA, EC 3.4.22.70) is a transpeptidase derived from Staphylococcus aureus
Fourier transform infrared spectroscopy (FTIR) was used to characterize chitosan particles, chitosan particles crosslinked with glutaraldehyde, and chitosan particles with immobilized SrtA
We have prepared chitosan macro-particles by the neutralization method and they were applied to Sortase A immobilization using glutaraldehyde as a crosslinking agent
Summary
Sortase A (SrtA, EC 3.4.22.70) is a transpeptidase derived from Staphylococcus aureus. It can recognize the Leu-Pro-X-Thr-Gly (LPXTG, X standing for any amino acid except cysteine) pentapeptide sequence at the C-terminus of protein and cleave the amide bond between threonine and glycine residues to form an acyl-enzyme complex, which subsequently reacts with nucleophiles containing oligo-glycine to generate a ligation product [1,2,3]. Enzyme SrtA is readily accessed by using recombinant expression technology in Escherichia coli [25]. It has been reported that the expression yield of SrtA in E. coli was optimized to a level of approximately 232 mg·L−1 [28], it was reported that the enzyme and substrates are used at a near-equimolar concentration in some cases.
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