| The Silkworm pupa protein is a premium natural protein.The hydrolysate prepared by various enzymatic hydrolysis technology has many efficacy like reduce blood pressure,anti-oxidation,anti-fatigue,anti-tumor and so on.It has a broad market prospect.Determine the amino acid composition of Silkworm Pupa,Silkworm Pupa protein hydrolysate having molecular weight>5 kDa(SN1),hydrolysate having molecular weight<5 kDa(SN2)using reversed-phase high performance liquid chromatography(RP-HPLC)with precolumn derivatization,meanwhile determined the ACE inhibitory activity.Experimental results show that Silkworm Pupa,Silkworm Pupa protein hydrolysate having molecular weight>5 kDa(SN1),hydrolysate having molecular weight<5 kDa(SN2)these three components in the full range of amino acids,and the ratio of content of essential amino acids and 18 amino acids are in line with the WHO/FAO reference protein pattern.Research the charge of amino acid composition after silkworm pupa protein hydrolysed with neutral protease and its affection to ACE inhibitory activity.The result indicate that the amount of hydrophobic amino acids may contribute to ACE inhibitory activity in SN2 is 49.15%,it was improved significantly compared to SN1 and Silkworm pupa protein values of 40.55%and 45.21%,respectively.The Pro and aromatic amino acids(Trp,Phe,Tyr)are major affected to ACE inhibitory activity in hydrophobic amino acids.The amount of Pro and aromatic amino acids in SN2 were increased to 7.75%and 19.20%compared to that of SN1(5.62%and 13.59%)and silkworm pupa protein(7.42%and 15.81%),the ACE inhibitory activity of SN2 increased to 47.6%in comparison with the SN1 and silkworm pupa protein values(Sample concentration is 0.20 mg·mL-1).It was suggest that the charge of hydrophobic amino acid composition reflect the variation trend of ACE inhibitory activity,and the main ACE inhibitory activity focus on SN2.The SN2 component with an Ion Exchange Chromatographic separation and purification,comprehensive survey of the experimental results of static adsorption and dynamic adsorption that determined the SN2 components separation conditions:6 g wet weight of D201 resin,the concentration of phosphate equilibration buffer is 0.01 moL·L-1(pH 8.5),the gradient concentration of NaCl eluate is 0?1.0 moL·L-1,sample volume of SN2 is 200 mg,the flow rate,the balance of liquid flow rate and elution flow all are 1.0 mL·min-1.In this separation conditions,the ACE inhibitory activity is mainly concentrated in the component penetrating peaks IE 1,the ACE inhibitory activity of IE1 is 47.6%(concentration is 0.008 mg·mL-1),has a significant blood pressure lowering effect.The IE1 component with an Gel Filtration Chromatography separated and purification,through a series of visits to the separation conditions that determined the final separation conditions:gel type selection Sephadex G-15,column specifications select 20 × 500 mm column,eluent selection deionized water,elution flow rate is 1.0 mL·min-1,sample volume is 200 mg.The eluted fraction is divided into a GF1,GF2 and GF3 three components in the separation condition,the ACE inhibitory activity is mainly concentrated in the GF2 component,the ACE inhibitory activity of GF2 is 69.28%(concentration is 0.008 mg·mL-1).The GF1,GF2 and GF3 component molecular weight measurement results showed that the different distribution of the polypeptide in respective components,the experimental reach the purpose of separation.The GF2 component was isolated by Reversed Phase Liquid Chromatography,after optimized the chromatographic separation conditions that determined the analysis chromatography conditions and preparative chromatography conditions for GF2 component separation conditions.The GF2 component after the first phase separation to obtain a maximum active ingredient HP6,it ACE inhibitory activity is 76.27%(concentration is 0.006 mg·mL-1).The HP6 component for the second liquid phase separation to give the highest active component HP64,it through Reversed Phase Liquid Chromatography analysis and High Performance Capillary Electrophoresis analysis can be identified as a single component,the IC50 value is 0.0013 mg·mL-1.HP64 has a significant ACE inhibitory effect,and achieve the purpose of the separation and purification antihypertensive peptides. |
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