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.

Keywords: ACE inhibition; Aspergillus niger derived prolyl endoproteinase; Bioactive peptides; Bovine β-casein; Food proteins; LC–MS; Substrate specificity.