Study On Prolyl Endopeptidase From The Muscleof Silver Carp(Hypophthalmichthys Molitrix) And Its Inhibitory Peptide

The physiological process of fish muscles is closely related to a variety of endogenous proteases.Prolyl endopeptidase?PEP?is an intracellular serine protease of S9 family,which cleaves short-length peptides?<33 amino acids?at the carboxyl side of an internal proline.In this study,silver carp was used as raw material to analyze its endogenous PEP.Target protease was isolated and purified by ammonium sulfate fractionation,column chromatographies including Q-Sepharose,Sephacryl S-200,Hi Trap^TM Phenyl HP and Hydroxyapatite.The results of SDS polyacrylamide gel electrophoresis?PAGE?revealed that the molecular mass of purified protease was about 80 kDa.There were two different subtypes of enzyme molecules on native-PAGE.The protease was positive for rabbit anti-tilapia PEP polyclonal antibody on Western blotting.Peptide mass fingerprinting identified thirteen fragements matching the PEP sequences of Danio rerio and Sinocyclocheilus rhinocerous with degrees of 100%and 98%,respectively,confirming that the purified protease was PEP.The circular dichroism analysis showed that the secondary structure of PEP was dominated by random coils?32.2%?,followed by?-sheet?27.4%?and?-helix?22.4%?,and with the lowest proportion of?-turn?18.2%?.The enzymatic analysis revealed that the optimum temperature of PEP was 35�C and the optimum pH was 6.0.Substrate specificity analysis indicated that PEP was able to specifically hydrolyze the Pro of the carboxyl side of fluorescent substrate.The study found that metal ions including Cu²⁺,Zn²⁺and Al³⁺,and inhibitor SUAM-14746 showed significant inhibitory effects on PEP activity.Besides,SUAM-14746 performed competitive reversible inhibition on PEP and the inhibitory activity(IC₅₀)was 0.67?mol/L.The enzyme catalytic kinetics study showed that the Michaelis constant?K_m?of PEP was 3.54?mol/L and the catalytic number(k_cat)was 16.9/s.PEP was expressed widely in brain,heart,liver,muscle,intestine,spleen and sputum.The expression of PEP was highest in brain tissue,followed by heart tissue,and the expression in other tissues was equivalent.PEP participates in the body metabolism of mammals by hydrolyzing protein peptides.Many studies found that the activity of PEP increased significantly in patients who suffered from neurodegenerative diseases.Thus,PEP has become a new target for disease treatment.Porphyra Haitanensis,marine red algae,is rich in protein,e.g R-phycoerythrin,it has multiple functions.The simulated gastrointestinal fluid digestion product of Porphyra haitanensis has PEP inhibitory activity.In this paper,R-phycoerythrin with relatively high purity(A₅₆₅/A₂₈₀ of 5.4)was obtained from Porphyra Haitanensis by ammonium sulfate fractionation and DEAE-Sepharose anion exchange chromatography.The purified R-phycoerythrin was hydrolyzed to prepare PEP inhibitory peptides with reference to the human digestion pattern of the food.Optimal conditions for enzymatic hydrolysis were performed at the ratio of pepsin to R-phycoerythrin of 0.5%?w/w?at pH 1.2,temperature of 37�C for 30 min,followed by digestion with 0.25%?w/w?trypsin and chymotrypsin at pH 7.5,temperature of 37�C for 30 min.A single active component of R-phycoerythrin hydrolysate was purified by continuously separation using Superdex^TM peptide10/300 GL gel column and ZORBAX Eclipse Plus C18 RP-HPLC column.Peptide mass fingerprinting of the active component identified six fragments matching the R-phycoerythrin beta chain with match degree 100%.Furthermore,two tripeptides PGL and IAP were obtained from MICEPGLIAP by predicting the digestive properties with pepsin and pancreatic enzyme.Molecular docking results showed that PGL was an uncompetitive inhibitory peptide and IAP was a competitive inhibitory peptide for PEP.In vitro activity verification revealed that the inhibitory activity(IC₅₀)of IAP on PEP was 1.49 mmol/L.This study explored in detail the enzymatic properties of PEP from freshwater silver carp and provided a theoretical reference for exploring the physiological functions of PEP in fish organisms.The preparation of PEP inhibitory peptides from R-phycoerythrin food-derived protein using enzymatic method provided technical support and theoretical reference for the preparation of bioactive peptides.