title: Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-casein and determination of ACE inhibitory activity.
abstract: The hydrolytic specificity of Aspergillus niger prolyl endoproteinase (An-PEP) on purified β-casein (β-CN) was assessed. This analysis confirmed cleavage at the C-terminal side of Pro residues. An-PEP also had the ability to cleave at the C-terminal side of Ala, Glu, Gly, Ser, Lys and Leu. Incubation of purified β-CN with An-PEP resulted in the generation of highly potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. The most potent hydrolysate was obtained after 24h incubation (ACE IC50=16.41±6.06μg/mL). Fourteen β-CN derived C-terminal Pro-containing di-, tri, and tetrapeptides which were predicted in silico to be released following An-PEP hydrolysis or which were detected by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) in the 24h hydrolysate were synthesised and characterised for their ACE inhibitory activity. The most potent inhibitory peptides were Ile-Gln-Ala (β-CN f187-189) and Val-Glu-Pro (β-CN f116-118) having ACE IC50 values of 32.9±9.2 and 63.7±12.0μM, respectively. The hydrolysates generated appear to have the most potent ACE IC50 values reported for a food derived hydrolysate to date.
authors.affiliation: Department of Life Sciences, University of Limerick, Ireland.
authors.forename: Roseanne
authors.initials: R
authors.lastname: Norris
authors.affiliation: Department of Life Sciences, University of Limerick, Ireland; Food for Health Ireland, University of Limerick, Ireland.
authors.forename: Alexey
authors.initials: A
authors.lastname: Poyarkov
authors.affiliation: Department of Life Sciences, University of Limerick, Ireland.
authors.forename: Martina B
authors.initials: MB
authors.lastname: O'Keeffe
authors.affiliation: Department of Life Sciences, University of Limerick, Ireland; Food for Health Ireland, University of Limerick, Ireland. Electronic address: dick.fitzgerald@ul.ie.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: FitzGerald
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology*; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D004150:Dipeptidases / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D007703:Peptidyl-Dipeptidase A / Q000032:analysis; D053719:Tandem Mass Spectrometry
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D002364:Caseins; D005656:Fungal Proteins; D010455:Peptides; D004150:Dipeptidases; C022454:prolyl dipeptidase; D007703:Peptidyl-Dipeptidase A
keywords: ACE inhibition; Aspergillus niger derived prolyl endoproteinase; Bioactive peptides; Bovine β-casein; Food proteins; LC–MS; Substrate specificity