鹿茸菇子实体新纤溶酶的高效分离纯化

Efficient isolation and purification of a novel fibrinolytic enzyme from fruiting bodies of Lyophyllum decastes

采用层析分离法从食药兼用真菌鹿茸菇(Lyophyllum decastes)的子实体中纯化得到了一种新纤溶酶,采用Edman降解法测定了该酶N-端氨基酸序列。结果显示,鹿茸菇子实体经干燥、粉碎、浸提后获得的含纤溶酶的粗酶液,依次经过盐析、Octyl-Sepharose Fast Flow疏水相互作用层析、SP-Sepharose High Performance离子交换层析和Source 15PHE疏水相互作用层析分离后获得单一组分的纤溶酶,酶比活力达到了4105.78 U/mg,纯化倍数为206.09,酶活力回收率为30.91%。Native-PAGE电泳和SDS-PAGE电泳检测结果均表明,该纤溶酶纯度达到了电泳纯,相对分子量为30.9 kDa。Edman降解法测定纤溶酶的N-端氨基酸序列为:Gly-Ala-Val-Thr-Gln-Cys-Asn-Ala-Pro-Trp-Gly-Leu,经过NCBI数据库比对,该纤溶酶为一种新纤溶酶。本文的研究为纤溶酶的研发提供了新的思路和方法。

A novel fibrinolytic enzyme was isolated and purified from the fruiting bodies of Lyophyllum decastes.The N-terminal amino acid sequence of the enzyme was determined by Edman degradation method.The fruiting bodies of Lyophyllum decastes was dried,crushed and extracted with phosphate solution to obtain crude enzyme solution.The crude enzyme solution was isolated and purified by using ammonium sulfate precipitation,Octyl-Sepharose Fast Flow hydrophobic chromatography,SP-Sepharose high performance ion chromatography and Source 15PHE hydrophobic chromatography to obtain a single component with fibrinolytic activity.The specific activity of the enzyme was 4105.78 U/mg,the purification fold was 206.09,and the recovery rate of the activity was 30.91%.Native-PAGE and SDS-PAGE showed that the fibrinolytic enzyme reached electrophoretic purity and the molecular weight was 30.9 kDa.The N-terminal sequences of the fibrinolytic enzyme were determined by the Edman degradation method,and the sequences was Gly-Ala-Val-Thr-Gln-Cys-Asn-Ala-Pro-Trp-Gly-Leu.By NCBI database comparison,it was found that the fibrinolytic enzyme was a novel fibrinolytic enzyme.The research provides a new idea and method for the research and development of fibrinolytic enzyme.