Effective separation of prolyl endopeptidase from Aspergillus Niger by aqueous two phase system and its characterization and application

Abstract

Aspergillus niger prolyl endopeptidase (An-PEP) has become a research focus because of its advantages in specifically cleaving the C-terminal peptide bond of proline residues, especially it was an industrial food-grade acidic PEP. Aqueous two-phase system (ATPS) was first applied for separating An-PEP from fermentation broth. Via response surface method (RSM) experiment, an effectively separation of An-PEP was achieved by ATPS containing27% (w/w) ethanol and 14.5% (w/w) (NH₄)₂SO₄ at pH 6.0 with the recovery of 90.29 ± 0.23% and purification coefficient of 15.35 ± 0.30. The purified An-PEP was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fourier transform infrared (FTIR) and fluorescence spectrometry. The optimum temperature and pH of An-PEP were 40 °C and 4.5-5.0, respectively. An-PEP was activated and stabilized by Ca²⁺ but inhibited by Fe³⁺. The enzymatic application of purified An-PEP was evaluated by hydrolyzing egg white protein (EWP) to prepare bioactive peptides. The obtained hydrolysates had good scavenging ability of OH and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, angiotensin converting enzyme (ACE) inhibitory activity and anti-gout activity. This research realized a low-cost, high-efficiency and simple separation technology of An-PEP and provided a broader idea for the preparation of bioactive peptides and the application of An-PEP.

Keywords: Aqueous two-phase system; Aspergillus niger; Bioactive peptides; Prolyl endopeptidase; Properties; Purification.