Isolation And Characterization Of ACE Inhibitory Peptides From Volutharpa Ampullacea Perryi Based On Affinity Chromatography

Hypertension is one of the main diseases and a common cardiovascular disease,which influence human body health.Angiotensin converting enzyme(ACE)is a key enzyme in the renin angiotensin system,which can promote the conversion of angiotensin I to angiotensin II leading to elevated blood pressure.Angiotensin converting enzyme inhibitor(ACEI)can inhibit the activity of angiotensin ?,which can achieve the purpose of lowering blood pressure.In this paper,the immobilized enzyme affinity separation method was used to separate and purify the ACE inhibitory peptides from the Volutharpa ampullacea perryi.It provide a new possibility for the development of natural healthy ACE inhibitory peptides without any side effects.The SBA-15 for immobilizing ACE was prepared.It was characterized by N2 sorption and XRD.Zn-SBA-15 was prepared by adsorbing Zn2+ on SBA-15.The crude extract ACE from lung was fixed to the microspheres by affinity adsorption technology.The feature of SBA-15 by a variety of characterization methods showed excellent porosity and specific surface area.The results show that the prepared SBA-15 can be used as a good carrier for the preparation of immobilized ACE.Through the optimization of single factor experiment,the optimum conditions for the immobilization of ACE was that protein concentration was 55 g/L,the temperature was 50 ?,pH was 8.3,reaction time was 60 min.The immobilized ACE activity was up to 0.3171 U/g.The optimum pH 8.3 of immobilized ACE was obtained by the experimental on the properties of the enzyme.The optimum temperature was 42 ?.The immobilized ACE obtained in this study has high activity and stability.The process of immobilized ACE was sample.It provides the basis for the research and application of separation and purification of ACE inhibitor peptides by immobilized ACE.The crosslinked CL 4B agarose beads was applied as carriers to immobilize ACE.The epichlorohydrin was used as activator for density modification of epoxy microspheres.The influencing factors of epoxy density in reaction were optimized.The reaction optimal conditions were that the NaOH concentration was 0.9 mol/L,NaBH4 concentration was 0.5 g/L,the volume fraction of epichlorohydrin was 40%,reaction temperature was 40 ?,reaction time was 2 h.The density of epoxy Sepharose surface activation was up to 113.52 mol/g.The crude ACE was immobilized onto activated agarose microspheres by covalent coupling method.The optimal conditions of immobilized ACE were determined by single factor experiments.The reaction optimal conditions were that the PH was 8.8,the reaction time was 1.5 h,the reaction temperature was 50?,the concentration of enzyme was 65 g/L.The enzyme activity of immobilized ACE reached the maximum of 0.1477 U/g.The process of obtaining immobilized ACE is simple and the immobilized ACE activity is good,which provides a good carrier for the separation and purification of ACE inhibitory peptides.A comparative analysis was done on the two kinds of immobilized enzyme preparation in the enzyme activity,nature and the simplicity of the experiment and the economic of raw material.The Zn-SBA-15 immobilized ACE is more suitable as affinity carrier to the separate and purify ACE inhibitory peptides.Finally,Zn-SBA-15 immobilized ACE was used as affinity carrier.The ACE inhibitory peptides were separated and purified by the affinity between the carrier and the peptides.The ACE inhibitory peptides bonded on the affinity carrier were released by increasing the salt concentration.Furthermore,the separation and purification of ACE inhibitory peptides were separated by Reversed Phase High Performance Liquid Chromatography.Two peptides sequences with ACE inhibitory were identified as Ile-Val-Thr-Asn-Trp-Asp-Asp-Met-Glu-Lys(IC50=2.08 mM)at 625.790(2+)m/z and Val-Gly-Pro-Ala-Gly-Arg-Pro-Gly(IC50=4.66 mM)at 355.6992(2+)m/z by Q-TOF(MS/MS).At the same time,the inhibition activity and inhibition type of the two peptides were determined,including the non competitive inhibition and mixed competitive inhibition of ACE,respectively.In this study,immobilized ACE-with high activity and stability was obtained by immobilization,and it could be used to separate and purify ACE inhibitory peptides from proteins and peptides from other sources.Our studies suggest that the Ile-Val-Thr-Asn-Trp-Asp-Asp-Met-Glu-Lys and Val-Gly-Pro-Ala-Gly-Arg-Pro-Gly from the Volutharpa ampullacea perryi protein hydrolysate were a potent ACE inhibitor and may be used to decrease blood pressure.In this study,immobilized ACE with high activity and stability was prepared by immobilization,which were Zn-SBA-15 immobilized ACE and CL 4B agarose immobilized ACE respectively.Zn-SBA-15 immobilized ACE was successful applied as a carrier in purifing ACEIP from Volutharpa ampullacea perryi by affinity chromatograph.The two ACE inhibitory peptides were isolated,which can be used as potential ACE inhibitors and to lower blood pressure.This study was providing a basis and possibility of potential in preparing ACEIP from other sources of protein and its hydrolysis product by affinity chromatograph in using immobilized ACE as affinity carrier which was prepared by this study.