One-step high efficiency separation of prolyl endopeptidase from Aspergillus niger and its application

Prolyl endopeptidase from Aspergillus niger (An-PEP) is an enzyme that recognizes C-terminal peptide bonds of amino acid chains and cleaves them by hydrolysis . An aqueous two-phase system (ATPS) was used to separate An-PEP from fermentation broth. Through single factor experiments, the ATPS containing 16 % ( w/w ) PEG2000 and 15 % ( w/w ) (NH 4 ) 2 SO 4 at pH 6.0 obtained the recovery of 79.74 ± 0.16 % and the purification coefficient of 7.64 ± 0.08. It was then used to produce soy protein isolate peptide (SPIP) by hydrolysis of soy protein isolate (SPI), and SPIP-Ferrous chelate (SPIP-Fe) was prepared with SPIP and Fe 2+ . The chelation conditions were optimized by RSM , as the chelation time was 30 min, chelation temperature was 25 °C, SPIP mass to VC mass was two to one and pH was 6.0. The obtained chelation rate was 82.56 ± 2.30 %. The change in the structures and functional features of SPIP before and after chelation were investigated. The FTIR and UV–Vis results indicated that the chelation of Fe 2+ and SPIP depended mainly on the formation of amide bonds. The fluorescence, SEM and amino acid composition analysis results indicated that Fe 2+ could induce and stabilize the surface conformation and change the amino acid distribution on the surfaces of SPIP. The chelation of SPIP and Fe 2+ resulted in the enhancement of radical scavenging activities and ACE inhibitory activities. This work provided a new perspective for the further development of peptide-Fe chelates for iron supplement.