Purification And Interaction Mechanism Of DPP-? Inhibitory Peptides From Pacific Oyster(Crassostrea Gigas)

Type II diabetes mellitus?T2DM?is a chronic metabolic disease caused by insulin resistance and its incidence rate is increasing annually.Glucagon-like peptide-1?GLP-1?and glucose-dependent insulin-stimulating polypeptides?GIP?play important roles in regulating the secretion of insulin and glucagon.However,GLP-1 and GIP can be degraded rapidly into inactive fragments by dipeptidyl peptidase-IV?DPP-IV,EC 3.4.14.5?,affecting the regulation of blood glucose.The effective inhibition of DPP-IV is one of the important means for the treatment of T2DM.In this study,DPP-IV was isolated and purified from pig kidney,and used as a tool enzyme to purify DPP-IV inhibitory peptides from oyster hydrolysate.The mechanism between the inhibitory peptide and DPP-IV was analyzed.DPP-IV was purified from pig kidney by acid precipitation,ammonium sulfate fractionation and gel filtration chromatography.The results showed that DPP-IV was a glycoprotein with a molecular weight of about 110 kDa,and its isoelectric point was about 6.1.Twenty peptide fragments with 296 amino acid residues were obtained,and the amino acid sequence was identical to DPP-IV from Sus scrofa.CD analysis showed that secondary structure of DPP-IV comprised?-helix?6.8%?,?-sheet?52.5%?,?-turn?16.6%?and random coils?29%?.The secondary structure of DPP-IV did not change when the temperature was below 60°C.With the increase of temperature,the percentage of?-helix,?-sheet and?-turn increased,while the random coils decreased.The activity of DPP-IV remained relatively high when the temperature was below 60°C and pH was between 4.0 and 10.0,indicating it has good thermal stability and pH stability.The denaturation temperature?T_d?of DPP-IV was 72.8±0.2°C.The intrinsic fluorescence spectrum of DPP-IV did not change when the temperature below 60°C while increased when it shifted to 70°C.The intrinsic fluorescence was relatively stable in the pH range of 5.0-9.0 and the fluorescence intensity decreasesd due to fluorescence quenching when pH is below 4.0 or higher than 10.0.The IC₅₀values of DPP-IV on Zn²⁺and Ca²⁺were 126.47±1.80?mol/L and 48.08±0.23 mmol/L,respectively,and did not change the secondary structure of DPP-IV.As the concentration of Zn²⁺and Ca²⁺increased,the fluorescence intensity of DPP-IV increased first and then decreased.DPP-IV was used as tool enzyme to prepare inhibitory peptides from Crassostrea gigas.It showed that the best inhibitory activity when pancreatin concentration was 0.8%hydrolyzed for90 min at pH 8.0.The 3 kDa ultrafiltration membrane,Sephadex G-15 gel chromatography and RP-HPLC were used to separate the enzymatic hydrolysates,two peptides,APSTM and ILAPPER,were obtained by mass spectrometry and BIOPEP-UWM digestion.In vitro analysis of DPP-IV inhibitory activity showed that IC₅₀ values of APSTM and ILAPPER were 354.81±20.00?mol/L and 16.98±1.29?mol/L,respectively.APSTM and ILAPPER showed good digestion stability after simulating the gastrointestinal digestion in vitro.APSTM could be degraded,while ILAPPER could not be degraded by DPP-IV in the inhibition experiments of DPP-IV.The inhibition mechanism of DPP-IV inhibitory peptides was further analyzed.The inhibition kinetics results showed that the inhibition types of APSTM and ILAPPER were competitive/non-competitive mixed inhibition and competitive inhibition,respectively.The melting temperature?T_m?of DPP-IV was increased by 1.83°C and 7.62°C with APSTM and ILAPPER as determined by differential scanning fluorometry?DSF?.Molecular docking investigation showed that the two inhibitory peptides formed hydrogen bonds with Arg125,Glu205,Glu206,Tyr662 in the S1 and S2 pocket of DPP-IV,and ILAPPER revealed a higher inhibitory activity due to formation of hydrogen bonds with Ser630 and His740 in the catalytic center.This study simplified the preparation method of DPP-IV and provided a tool enzyme for the separation of DPP-IV inhibitory peptides from oyster.The study provides a theoretical basis for the development and application of marine functional food.