National Engineering Laboratory for Cereal Fermentation Technology, State Key Laboratory of Food Science and Technology, School of Food Science and Technology , Jiangnan University , 1800 Lihu Road , Wuxi , Jiangsu 214122 , People's Republic of China.
College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing , Nanjing University of Finance and Economics , Number 3 Wenyuan Road , Nanjing , Jiangsu 210023 , People's Republic of China.
J Agric Food Chem. 2019 Apr 3;67(13):3679-3690. doi: 10.1021/acs.jafc.9b01069. Epub 2019 Mar 18.
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 (IC = 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.
先前报道的油菜籽（ Brassica napus ） napin 衍生肽在计算机上显示可抑制 DPP-IV。在本研究中，从油菜籽中提取的 napin 用商业酶水解，并用超滤膜过滤。然后，通过 Sephadex G-15 凝胶过滤柱和制备型 RP-HPLC 对 napin 水解产物进行纯化。从酶解产物的 DPP-IV 抑制活性（IC = 0.68mg/mL）来看，复合酶 alcalase 和胰蛋白酶的双酶组合方法是最有利的。使用 HPLC-三重-TOF-MS/MS 鉴定了三种肽和一种修饰肽（N 端的焦谷氨酸突变）。还确定了 DPP-IV 抑制活性和酶抑制类型。同时，通过分子对接研究了与所选肽与 DPP-IV 相互作用相关的关键残基。IPQVS 具有与 Diprotin A 一致的关键氨基酸残基（Tyr547、Glu205 和 Glu206）。ELHQEEPL 可以与 DPP-IV 的 S3 口袋中的 Arg358 形成更好的共价键。
