Aspergillus niger derived prolyl endoproteinase
Substrate specificity
Bovine beta-casein
ACE inhibition
LC-MS
Bioactive peptides
Food proteins
ANGIOTENSIN-CONVERTING ENZYME
SPONTANEOUSLY HYPERTENSIVE-RATS
ANTIHYPERTENSIVE PEPTIDES
STRUCTURAL-ANALYSIS
WHEY PROTEINS
MILK
PURIFICATION
IDENTIFICATION
SEQUENCE
CHEESE
The hydrolytic specificity of Aspergillus niger prolyl endoproteinase (An-PEP) on purified beta-casein (beta-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 beta-CN with An-PEP resulted in the generation of highly potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. The most potent hydrolysate was obtained after 24 h incubation (ACE IC50 = 16.41 +/- 6.06 - mu g/mL). Fourteen beta-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 24 h hydrolysate were synthesised and characterised for their ACE inhibitory activity. The most potent inhibitory peptides were lie-Gin-Ala (beta-CN f187-189) and Val-Glu-Pro (beta-CN f116-118) having ACE IC50 values of 32.9 +/- 9.2 and 63.7 +/- 12.0 mu M, respectively. The hydrolysates generated appear to have the most potent ACE IC50 values reported for a food derived hydrolysate to date. (C) 2014 Elsevier Ltd. All rights reserved.