亚麻种子中胰蛋白酶抑制剂的分离纯化及性质研究

陈颖璐; 石亚伟

doi:10.13386/j.issn1002-0306.2016.22.037

亚麻种子中胰蛋白酶抑制剂的分离纯化及性质研究

Purfication and properies of the trypsin inhibitor from flax seeds

摘要

摘要: 将亚麻种子去壳粉碎,经丙酮脱脂和Tris-HCl缓冲液提取后,Q-Sepharose^TM Fast flow离子交换层析一步纯化,获得电泳纯的亚麻胰蛋白酶抑制剂（LUTI）,纯化倍数可达9.62,活力回收率为6.25%,比活力为55.35 U/mg,SDSPAGE电泳显示LUTI分子量大小约为8 ku。质谱鉴定属于Potato型胰蛋白酶抑制剂,其抑制活性在p H2.0⁶.0以及70℃以下有较好的稳定性,最适p H为6.0,最适温度为40℃,属于一种非竞争性抑制剂,Ki值为9.18×10^-4mol/L。DTNB法检测LUTI含有一对二硫键,二硫键存在有助于提高LUTI的稳定性和活性。

Abstract: The Linum usitatissimum trypsin inhibitor( LUTI) had been isolated from naked flax seeds by acetone fractionation,Tris- HCl buffer extraction and Q- Sepharose~(TM) Fast flow.With the purification steps mentioned above,the overall recovery of enzymatic activity of 6.25%,the specific activity of 55.35 U / mg and the purification fold of9.62 for LUTI from crude extraction was achieved.The LC- ESI- MS showed LUTI belonged to Potato trypsin inhibitor family and the relative molecular weight was 8 ku by SDS- PAGE.The trypsin inhibitory activity of LUTI was stable below 70 ℃,as well as p H2.0~ 6.0. The optimum temperature of LUTI was 40 ℃ and the optimum p H was6.0.LUTI was a non- competitive inhibitor by kinetic assay with an inhibition constant Kiof 9.18 × 10~(-4)mol / L and contained a pair of disulfide bond by DTNB assay,which was related with the stability and activity of LUTI.