珍珠蚌肉二肽基肽酶-IV抑制肽的制备与分离

Preparation and Purification of Dipeptidyl Peptidase-IV Inhibitory Peptides from Pearl Mussel Muscle

目的:以珍珠蚌肉为原料制备对二肽基肽酶(dipeptidyl peptidase,DPP)-IV具有抑制作用的生物活性肽。方法:利用两步酶解法并优化珍珠蚌肉酶解条件,筛选体外DPP-IV抑制活性较高组分进行超滤膜浓缩,Superdex^(TM) peptide 10/300 GL凝胶柱层析与反相高效液相色谱(reverse phase-high performance liquid chromatography,RP-HPLC)技术分离纯化出酶解液中的DPP-IV抑制肽,最终通过液相色谱-串联质谱鉴定其氨基酸序列。结果:中性蛋白酶与碱性蛋白酶对珍珠蚌肉均有良好降解效果,经3 kDa超滤膜浓缩后,酶解产物的DPP-IV抑制率可达67.4%。通过凝胶过滤柱和RP-HPLC进一步分离纯化出6个活性较高的肽段,序列分别为FNAPAM、FIPNY、IYNPPTPF、LAMPYP、FFVVMP和LAGMP。进一步,以分子对接的方式分析6个肽段与DPP-IV的相互作用关系。结论:本研究利用珍珠蚌肉制备DPP-IV抑制肽,可为以水产加工副产物为原料制备功能性食品提供理论参考。

Objective:Bioactive peptides with an inhibitory effect on dipeptidyl peptidase-IV(DPP-IV)were prepared from pearl mussel muscle.Methods:Two-step enzymatic hydrolysis was adopted and enzymatic hydrolysis conditions were TM optimized.Fractions with in vitro DPP-IV inhibitory activity were concentrated by ultrafiltration,and purified by Superdex peptide 10/300 GL gel filtration column chromatography and reverse phase-high performance liquid chromatography(RP-HPLC).DPP-IV inhibitory peptides were purified from the enzymatic hydrolysate and their amino acid sequences were identified by liquid chromatography-tandem mass spectrometry.Results:Both neutral protease and alkaline protease could effectively hydrolyze pearl mussel muscle.After concentration using a 3 kDa cut-off ultrafiltration membrane,the DPP-IV inhibition rate of the enzymatic hydrolysate reached 67.4%.After separation by gel filtration column and purification by RP-HPLC,6 active peptides were obtained,whose sequences were FNAPAM,FIPNY,IYNPPTPF,LAMPYP,FFVVMP and LAGMP,respectively.Furthermore,the interactions between these peptides and DPP-IV were analyzed by molecular docking.Conclusion:Our present study provides a theoretical reference for the effective utilization of fish processing byproducts as raw materials for functional food production.