单环刺螠纤溶酶UFE-Ⅱ的分离纯化及其酶学性质

Separation,Purification and Zymological Property of Fibrinolytic Enzyme UFE-Ⅱ from Urechis unicinctus

目的分离纯化单一组分的单环刺螠纤溶酶UFE-Ⅱ,并分析其酶学性质。方法单环刺螠体腔液经离心、超滤、离子交换层析、凝胶过滤等方法进行分离纯化,得到单一洗脱峰酶组分,经Native-PAGE、SDS-PAGE和飞行质谱分析,测定其纯度和相对分子质量;并以酪蛋白为底物,Folin-酚试剂法测定酶活力,对其酶学性质进行分析。结果分离纯化的UFE-Ⅱ具有水解纤维蛋白的活性,其水解酪蛋白的比活力达到375.6U/mg,纯化倍数为10.3倍,回收率为14.0%。UFE-Ⅱ为单一组分,纯度达99%以上,相对分子质量为24329。UFE-Ⅱ的最适反应温度约为45℃;最适反应pH值为7.0;Ca2+、Mn2+和Fe2+是该酶的强激活剂;Fe3+、Cu2+和Pb2+对该酶活力具有一定的抑制作用;SBTI和PMSF能完全抑制酶活力,表明该酶为丝氨酸蛋白酶;糜蛋白酶抑制剂可部分抑制酶活力,亮抑酶肽、抑蛋白酶肽、苯甲脒可较弱地抑制酶活力。结论从单环刺螠体内成功分离纯化出纤溶酶UFE-Ⅱ,并分析了其酶学性质,该酶具有进一步开发利用价值。

Objective To separate and purify fibrinolytic enzyme UFE-Ⅱwith a single component from Urechis unicinctus and analyze its zymological property.Methods UFE-Ⅱ was purified from the coelomic fluid of Urechis unicinctus by centrifugation,ultrafiltration,ion exchange chromatography and gel filtration,determined for purity and relative molecular mass by Native-PAGE,SDS-PAGE and Q-Tof-MS,for enzyme activity with Folin-phenol reagent using casein as a substrate,then analyzed for zymological property.Results The purified UFE-Ⅱ showed activity in hydrolysis of fibrin.The specific activity in hydrolysis of casein,purification fold and recovery rate of the purified UFE-Ⅱ were 375.6 U /mg,10.3 and 14.0% respectively.UFE-Ⅱ was a single component with a purity of more than 99% and a relative molecular mass of 24 329.The optimal reaction temperature and pH value of UFE-Ⅱ were 45℃ and 7.0 respectively.Ca2＋,Mn2＋ and Fe2＋ were strong activators,while Fe3＋,Cu2＋ and Pb2＋ showed certain inhibitory effect on the enzyme activity of UFE-Ⅱ.SBTI and PMSF completely inhibited the activity of UFE-Ⅱ,indicating that UFE-Ⅱ was a serine protease.Chymotrypsin inhibitor showed partial inhibitory effect,while leupeptin,aprotinin and benzamidine showed weak inhibitory effect on the enzyme activity of UFE-Ⅱ.Conclusion UFE-Ⅱ was successfully separated and purified from Urechis unicinctus and analyzed for zymological property,which was worthy of further development.