DA201-C大孔吸附树脂对乳铁蛋白抗菌肽的分离纯化

Separation and purification of antimicrobial peptides from lactoferrin by DA201-C macroporous adsorbent resin

该研究以吸附量、树脂动态吸附和不同乙醇浓度(体积分数)梯度洗脱为考察指标,优化了DA201-C大孔树脂对超滤处理后的乳铁蛋白抗菌肽溶液(分子质量<3000 Da)分离纯化的工艺条件。静态吸附结果表明,当树脂吸附12 h后,吸附量为479.7 mg/g、吸附率为95.94%;对吸附饱和的树脂使用不同浓度的乙醇静态解吸,结果显示,75%(体积分数)乙醇解吸率达到95.35%。该研究还确定了最优吸附条件:NaCl浓度0.6 mol/L、上样流速1.5 mL/min和上样质量浓度20 mg/mL。以大肠杆菌和金黄色葡萄球菌抑菌率为指标,用不同浓度乙醇对乳铁蛋白抗菌肽进行梯度洗脱,结果表明,75%乙醇洗脱组分抑菌活性最强,对大肠杆菌和金黄色葡萄球菌的抑菌率分别达到80.95%和74.42%,与未纯化前相比提高了12.2%左右。对不同洗脱组分的氨基酸分析,结果显示,抗菌肽的疏水性及正电荷性越大,抗菌活性越强。

In this study,the separation and purification conditions of lactoferrin antimicrobial peptide solution(molecular weight<3000)treated by ultrafiltration with DA201-C macroporous resin were optimized by using adsorption capacity,resin dynamic adsorption and gradient elution of different ethanol concentrations as indexes.Static adsorption results showed that the adsorption capacity was 479.7 mg/g and the adsorption rate was 95.94%after 12 h of resin adsorption.Static desorption of saturated resin with different concen-trations of ethanol showed that the desorption rate of 75%(volume fraction)ethanol reached 95.35%.The optimal adsorption conditions were determined as follows:NaCl concentration 0.6 mol/L,loading flow rate 1.5 mL/min and loading mass concentration 20 mg/mL.The gradient elution of lactoferrin antimicrobial peptides with different concentrations of ethanol was used as the index.The results showed that 75%ethanol eluted fraction had the strongest antibacterial activity against E.coli and Staphylococcus aureus,and the antibacterial rates of E.coli and Staphylococcus aureus reached 80.95%and 74.42%,respectively,which increased about 12.2%compared with be-fore purification.Amino acid analysis of different elution components showed that the greater hydrophobicity and positive charge of the an-timicrobial peptides,the stronger antibacterial activity.