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Identification and Quantification of DPP-IV-Inhibitory Peptides from Hydrolyzed-Rapeseed-Protein-Derived Napin with Analysis of the Interactions between Key Residues and Protein Domains

Xu, Feiran, Yao, Yijun, Xu, Xiaoying, Wang, Mei, Pan, Mengmeng, Ji, Shengyang, Wu, Jin, Jiang, Donglei, Ju, Xingrong, Wang, Lifeng
Journal of agricultural and food chemistry 2019 v.67 no.13 pp. 3679-3690
Brassica napus, amino acids, chemical bonding, computer simulation, enzyme inhibition, gel chromatography, hydrolysates, inhibitory concentration 50, mutation, peptides, protein domains, rapeseed, reversed-phase high performance liquid chromatography, subtilisin, tandem mass spectrometry, trypsin, ultrafiltration
Previously reported peptides derived from napin of rapeseed (Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IV-inhibitory activity (IC₅₀ = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.