| Antihypertensive peptides (ACE inhibitory peptides) from food protein sources can be used as functional food and replace some of anti-hypertension drugs for advantages of durable, safety and non side effects. Combining magnetic separation technology with the traditional separation method can realize the rapid purification of ACE inhibitory peptides. In this research, magnetic agarose microspheres were prepared by reverse-phase microemulsion method with agarose and Fe3O4 as the material. The microspheres were used for the preparation of magnetic affinity medium to separate ACE inhibitory peptides from Saurida elongate hydrolysate. After further purification by RP-HPLC, a new ACE inhibitory peptide was identified, and its inhibition mechanism was preliminarily studied. The main experimental results are as follows:(1) Magnetic agarose microspheres (MAMs) were prepared by reverse-phase microemulsion method. MAMs were prepared by reverse-phase microemulsion method with agarose and Fe3O4 the material. The preparation conditions were optimized by single factor experiment using the microsphere diameter as criterion. Scanning electron microscopy (SEM), infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and vibrating sample magnetometer (VSM) were used to study the properties of MAMs. The results showed that MAMs with an average size of 10.69 μm were obtained under following conditions:agarose concentration of 25.0 g·L-1, the water/oil ratio (W/O) of 1:10, concentration of Span 80 of 40.0 g·L-1, emulsifying temperature for 50 ℃, stirring rate of 900 r·min-1 and Fe3O4 concentration of 4.5 g·L-1. The microspheres showed good dispersion and were superparamagnetic, the saturation magnetization of MAMs was 5.67 emu·g-1 with the Fe3O4 content of 29.5%. It suggested that the rapid solid-liquid separation could carry out in the presence of an external magnetic field.(2) MAMs were activated by epichlorohydrin (ECH). Taking epoxy density as critorion, the optimal conditions of activation were determined by the single factor experiment. The epoxy density achieved maximum of 123.96 μmol·g-1 under the optimal conditions:0.9 mol·L-1 NaOH, ECH 40.0%(v/v),0.5 g·L-1 NaBH4;, temperature for 40℃ and the reaction time was 4 h.(3) Zn2+ was fixed to the activated MAMs to prepared Zn2+-chelated magnetic agarose microspheres (MAMs-IDA-Zn2+) using IDA as space arm. With chelating capacity of Zn2+ as critorion, the preparation conditions were optimized as follows:the IDA bonding time of 8 h, concentration of IDA of 0.8 mol·L-1, Zn2+ chelating time of 60 min, the concentration of Zn2+ of 0.03 mol·L-1. The maximum value of Zn2+ chelating capacity was 101.69 μmol·g-1.(4) The pig lung ACE was innovatively separated by magnetic immobilized metal affinity chromatography. Taking protein adsorption as critorion, the optimal conditions of adsorption were confirmed by the single factor experiment. The results showed that all ACE adsorbed to MAMs-IDA-Zn2+ and the maximum ACE specific activity of bound protein 0.037 U·mg-1 was achieved under the following conditions:the protein concentration of 3.0 mg·mL-1, NaCl concentration of 0.5 mol·L-1, the contact time of 20 min, pH 8.3. The loaded ACE were eluted by 1.0 mol·L-1 NH4Cl in the condition of pH 7.8 and elution time of 40 min. The ACE were purified to 14.3-fold with specific activity of 0.160 U·mg-1 and activity recovery of 65.8%.(5) The pig lung ACE extract was covalently immobilized on activated MAMs to prepare magnetic immobilized ACE (MAMs-ACE). With specific activity of MAMs-ACE as critorion, the preparation conditions were optimized by the single factor experiment. The specific activity of MAMs-ACE achieved maximum 0.128 U·g-1 under the optimal conditions:the protein concentration of 8.0 mg·mL-1, immobilized time of 2 h, the temperature 50℃ and pH 7.8. The optimum temperature and pH values of MAMs-ACE were determined as 42℃ and 7.8, respectively. MAMs-ACE has excellent pH stability and thermostability as compared to that of the free form. The immobilized enzyme retained 53% activity up to 10 cycles. After preserving in-20℃for 30 days, MAMs-ACE still kept 90.3% of the enzyme activity, and free enzyme retained 43.0% only, it suggested that immobilized ACE has better storage stability. The values of the Michaels constants Km for MAMs-ACE was smaller than free ACE, indicating immobilization of ACE could arise the affinity between ACE and substrate.(6) Adsorption performance of MAMs-ACE was studied. The specific adsorption properties of MAMs-ACE were determined using the ACE inhibitory peptide HLPLP as template. The adsorption conditions were optimised as follows:4.0 mg·mL-1 HLPLP, adsorption time was 30 min,0.3 mol·L-1 NaCl, pH 7.8. The specific adsorption yield of 22.11 mg·g-1 were obtained. Under the same experimental conditions, the maximum non-specific adsorption of MAMs-ACE was determined to be 2.46 mg·g-1 with bovine albumin (BSA) as the template. It suggested that MAMs-ACE could be used for the separation of ACE inhibitory peptide for the excellent specificity adsorption.(7) ACE inhibitory peptides were separated from Saurida elongate hydrolysate by MAMs-ACE. The adsorption conditions were optimized through single factor experiments. The maximum adsorption capacity of MAMs-ACE achived 29.50 mg·g-1 under the following conditions: the protein concentration of 8.0 mg·mL-1, the adsorption time was 45 min, the concentration of NaCl of 0.2 mol·L-1 and pH7.8.90% of loaded protein were eluted by 2.0 mol·L-1 NaCl for 8 times and 30min each time. The adsorption capacity of MAMs-ACE decreased with the increase of repeated use, and the immobilized ACE could be re-used 4 times at most. The affinity separated peptides were further purified by RP-HPLC to obtain a new ACE inhibitory peptide. The peptide was analyzed by mass spectrometer, and the amino acid sequence was:Gly-Met-Lys-Cys-Ala-Phe (GMKCAF), its IC5o was 45.7 μmol·L-1.(8) The inhibition type of GMKCAF (GF-6) was determined by Lineweaver-Buck method, the result showed that GF-6 belongs to the non competitive inhibitor. Using initial rate method and isothermal titration calorimetry (ITC) to determine Km of ACE, and the Km values were 0.327 mmol·L-1 and 0.347 mmol-L"1, respectively. It suggested that the ITC measured result was credible.(9) Thermodynamics of inhibition of captopril and GF-6 were studied by ITC. Thermodynamic parameters of captopril and GF-6 with ACE were determined by ITC experiments:Ka, n, AH, ACP and AG The results showed that the combinations of captopril and GF-6 with ACE were both spontaneous endothermic process. Stoichiometry for the binding of captopril and GF-6 to ACE were 1:1 and 40:1, respectively. It verified their inhibition type. The thermodynamic research indicated that associations captopril-ACE and GF-6-ACE have are governed by entropic contribution with enthalpy-entropy compensation at all temperatures examined. |
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