Study On Preparation And Inhibition Mechanism Of Dipeptidyl Peptidase Ⅳ Inhibitory Peptide Derived From Yeast

Diabetes is a chronic metabolic disease characterized by hyperglycemia,which seriously affects human physical and mental health.In recent years,the dipeptidyl peptidase Ⅳ（DPP-Ⅳ）inhibitory peptide,which has a good regulation and control effect on hyperglycemia,has attracted much attention.Yeast is rich in protein,which is a microbial protein resource with great development value.In this study,enzyme that prepared by Bacillus subtilis fermentation have been used to hydrolysis yeast,then the separation,enrichment,screening and identification of DPP-Ⅳinhibitory peptides were realized.Besides these,enzyme inhibition kinetics and molecular docking were used to assess the molecular mechanism between the peptides and DPP-Ⅳ,finally the stability of DPP-Ⅳinhibitory peptide and its combined action with drugs were analyzed.The main research contents are as follows:（1）The growth and metabolic characteristics of Bacillus subtilis HU528 were analyzed,and it was found that the fermentation type of protease produced by HU528 belonged to non-growth coupling type.The fermentation conditions of HU528 in a 5 L fermentation tank were optimized,and the protease activity of the fermentation liquid was up to 4228.64 U/m L,which was 6.5 times that of the shaking flask fermentation.The batch fermentation was further investigated.Add 200 ml 0.05 g/m L glucose at constant speed for 20-40 h of fermentation significantly increased the fermentation volume and protease activity,and the highest enzyme activity reached 8189.22 U/m L,which was 93.66%higher than that without feeding.（2）The crude enzyme fluid of Bacillus subtilis was used for enzymatic hydrolysis of yeast.The hydrolysate with 4.5 h enzymatic hydrolysis had the highest inhibitory activity of DPP-Ⅳ,and the half-maximal inhibitory concentration(IC₅₀)was 0.84 mg/m L.The IC₅₀ of the highest active component F₂ was 0.41 mg/m L,which was significantly lower than that of the unseparated component.A total of 186 peptides were identified from F₂ by mass spectrometry,and nine peptides with potential DPP-Ⅳ inhibitory activity were screened out by using the online tool and DPP-Ⅳinhibitory peptide characteristics.（3）Five synthetic peptides were found to have high inhibitory activity against DPP-Ⅳby enzymatic activity inhibition assay,in which tetrapeptide WPLP had the highest DPP-Ⅳinhibitory activity with IC₅₀ of 178.90μmol/L.The inhibition of DPP-Ⅳ by five polypeptide compounds was investigated by enzymatic inhibition kinetics.WPLP was uncompetitive inhibition.Molecular docking showed that all five inhibitory peptides could spontaneously bind to DPP-Ⅳ.The inhibitory effects of different polypeptide compounds on DPP-Ⅳ were analyzed.It was found that different inhibitory types of DPP-Ⅳ inhibitory peptides had superposition effects at low concentrations.（4）The stability of DPP-Ⅳ inhibitory peptide WPLP was investigated.It was found that WPLP had thermal stability and p H stability,and could resist the hydrolysis of DPP-Ⅳ.WPLP was degraded after simulated gastric digestion,and the inhibitory activity of DPP-Ⅳ was reduced by nearly 40%.The combined inhibition of WPLP and sitagliptin was further analyzed by equivalent dose method.Low dose concentration of sitagliptin（30%inhibition rate）had a certain synergistic effect with WPLP,while the combined inhibition of medium and high dose concentration of sitagliptin（50%and 70%inhibition rates）with WPLP showed a superposition effect.