Co-expression Of Recombinant ACE Inhibitory Peptides With Antioxidant Peptides And Combined Antihypertensive Effects

Hypertension is the main cause of death of cardiovascular and cerebrovascular diseases,and it is a serious threat to human health.The food-borne ACE inhibitory peptides and antioxidant peptides,which have the advantages of mild effect,high safety and others,can play a role in lowering blood pressure by acting on different blood pressure regulating system and it become the research hotspot.The method of separation and extraction of active peptides from natural proteolytic products is complicated,low in production and high in cost,which limits the industrial application of active peptides.In this paper,a variety of ACE inhibitory peptides and antioxidant peptides were prepared in genetically engineered bacteria combination with high-density fermentation technology,to develop the compound peptides products which can combined antihypertensive effect based on different mechanism.The main research contents were as follows:designing active peptide multimers,constructing recombinant strains;optimizing fusion protein expression conditions;purifying fusion proteins and active peptide multimers;studying the in vivo and vitrol antihypertensive effects of enzymatic oligopeptides of active peptide multimers.The active peptide multimers were designed and the genetically engineered bacteria were constructed.Based on the bioinformatics analysis of the structure of active peptides,the enzymatic properties of gastrointestinal enzymes and the expression of peptides,the ACE inhibitors such as IVY and anti-oxidant peptides such as DTHK were assembled into active peptides mutilmer that could be activated by gastrointestinal enzymes.constructed E.coli BL21(DE3)/pET28a-6×His-SUMO-BPP-1,which was capable of expressing and isolating BPP-1.The results showed that the fusion protein 6×His-SUMO-BPP-1 was highly expressed in the form of inclusion bodies,and the expression level was about 53.64%of the whole cell protein after incubation for 5 hours at 37? and 1.0 mmol/L IPTG.The culture conditions of shake cells and fermentor were optimized.The optimum culture conditions were as follows:37?,4 ml/L glycerol,induced after cultured for 8 h,0.6 mmol/L IPTG,incubation for 14h.Under these conditions,the OD600 and dry weight of engineering bacteria reached 16.0 and 6.1 g/L,the expression level was about 59.09%of the whole cell protein.The effect of batch fermentation,constant speed flow and ferrmentation and exponential flowering on the fermentation of engineering bacteria was studied by using 3L fermentor.The results showed that the index fermentation was the best fermentation method.Controlling the constant pH and dissolved oxygen,the exponential feeding can get a higher amount of bacteria,the OD600 and dry weight of engineering bacteria reached 65.0 and 24.8 g/L,the expression level was about 63.46%of the whole cell protein.The preparation conditions of fusion protein and active peptide multimer were optimized.Through the inclusion body washing,dissolution and affinity purification,from the 1L engineering bacteria fermentation broth to get 1.93 g fusion protein,purity of 94.88%.Further,by using SUMO protease digestion and affinity purification,a purity of about 98.05%of the multirmer BPP-1 was obtained with a yield of 91.06%and a yield of 0.564 g/L.The results of trypsin digestion and UPLC-MADIL-TOF-TOF mass spectrometry showed that the multimer BPP-1 was correctly expressed.After digested with BPP-1,its enzymatic hydrolysis of the oligopeptide showed strong ACE inhibitory activity(IC50=0.004 mg/mL)and antioxidant activity,indicating that the active peptide in BPP-1 could be efficient release by gastrointestinal enzymes.The in vivo bioactivity of BPP-1-cleaved oligopeptides was studied.The effects of BPP-1 on the cell viability,NO,ET-1,MDA,ROS and apoptosis of EA.hy926 cells induced by H2O2 were studied by CCK-8 method,ELISA,fluorescence probe and flow cytometry.The results showed that H2O2 could induce the apoptosis and oxidative damage of EA.hy926 cells,while the enzymolysis of oligopeptide could significantly increase the cell viability and decrease the apoptosis rate,and dose-effect.The content of contraction factor ET-1 was significantly decreased,Increase the release of diastolic factor NO;can significantly reduce the MDA content,reduce intracellular ROS production.Studies have shown that BPP-1 enzymatic hydrolysis of oligopeptides on vascular endothelial cell activity,cell function and antioxidant stress system has a significant regulatory role,with a strong antihypertensive effect.The results of the above studies show that BPP-1 can be used as a terminal product for the prevention and treatment of hypertension after in vitro digestion,as well as the feasibility of batch preparation of biologically active peptides by genetic engineering bacteria.