pH-区带精制逆流色谱分离高F值寡肽新技术研究

Separation of Oligopeptides with High Fisher's Ratio by pH-Zone-Refining Counter-Current Chromatography

高F值寡肽是一种由2～8个氨基酸组成的生理活性肽。本研究采用胃蛋白酶和O／一胰凝乳蛋白酶对草鱼蛋白进行了两步酶解，高效液相色谱对酶解液进行了肽谱分析，以制备改善肝性脑病的高F值寡肽。结果表明，草鱼蛋白酶解液中含有高支低芳氨基酸的寡肽组分，开发一种pH一区带精制逆流色谱分离新技术，所用体系为甲基叔丁基醚：正丁醇：乙腈：水（2：2：1：5，V／V／V／V），上相中加入20mM三乙胺和20％磷酸二（2-乙基己基）酯（DEHPA）作为固定相，下相分两部分，分别加入10mM HCl和20m MHCl作为流动相，进行梯度洗脱。成功地对酶解液中的混合组分进行了分离，得到了5个高F值（F〉20）的分离组分，估测其F值（F=OD214nm／OD280nm）高达24．7—36．4。该结果为利用低值鱼蛋白酶解制备功能性活性肽提供了新的制备方法。

The oligopeptides with high Fischer＇ s ratio was made up of 2-8 amino acid compositions of hioactive peptides. A two-step hydrolysis for preparing the high Fischer value＇ s oligopeptides from fish protein of Ctenopharyngodon idellus by pepsin and a-chymotrypsin was investigated in this study. The peptide graphs of hydrolyzate were measured by high performance liquid chromatography. It was showed that the oligopeptides in the hydrolyzate were with high level of branched chain amino acids and low level of aromatic amino acids. A new preparation chromatographic technique, pH- zone-refining counter-current chromatography was developed, separation was performed with a two-phase solvent system composed of MtBE-n-butanol-CH3 CN-water （ 2： 2：1 ： 5, V/V/V/V） , 20 mM triethylamine and 20% percents of di- （ 2- ethylhexyl） phosphoric acid in organic phase ,the aqueous mobile phase was separated into two parts：10 mM hydrochloric acid in the first part and 20 mM hydrochloric acid in the second part, elute with gradient. Then the mixture components of hydrolyzate were separated successfully. Five components with high Fisher＇ s ratio （ F 〉 20 ） were found. Their F ratios（ F = OD214nm/OD280nm ）could reach up to 24.7-36.4. The research may be useful for the study and preparation of bioaetive peptide.