茶树菇子实体新纤溶酶的纯化及其溶栓抗凝性质研究  

Purification and fibrinolytic/anticoagulant activity of a novel fibrinolyticenzyme from the fruiting bodies of Agrocybe aegerita

作　　者：李冠龙[1,3] 刘晓兰 郑喜群[1,2] LI Guanlong;LIU Xiaolan;ZHENG Xiqun(College of Food and Biological Engineering,Qiqihar University,Qiqihar 161006,China;College of Food,Bayi Agricultural University,Daqing 163319,China;Key Laboratory of Corn Deep Proessing Theory and Techology,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔大学食品与生物工程学院,黑龙江齐齐哈尔161006 [2]黑龙江八一农垦大学食品学院,黑龙江大庆163319 [3]黑龙江省玉米深加工理论与技术重点实验室,黑龙江齐齐哈尔161006

出　　处：《食品与发酵工业》2020年第11期105-111,共7页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31171744);齐齐哈尔大学研究生创新科研项目(YJSCX2018-ZD16);黑龙江省自然科学基金重点项目(ZD201305);黑龙江省教育厅科研创新团队项目(135309113)。

摘　　要：从茶树菇子实体中提取、纯化纤溶酶,研究其体外的溶栓活性和抗凝活性,用于开发预防具有血栓倾向的心脑血管疾病的功能食品。对茶树菇(Agrocybe aegerita)子实体含有的纤溶酶进行研究,将子实体干燥、粉碎、生理盐水浸提,获得粗酶液,再依次经盐析、Sephadex G-25凝胶色谱、CM-Sepharose FF离子交换色谱、Source 15PHE疏水色谱和Mono S离子交换色谱纯化,最终得到纤溶酶(ACase)。该酶比活力为1731.05 U/mg,纯化倍数为246.94。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gelelectrophoresis,SDS-PAGE)和Native-PAGE电泳分析显示,ACase为二聚体蛋白,分子质量分别为31.4 kDa和21.2 kDa。Edman降解法分析显示,2个亚基N-端序列分别为AIVTQTNAPWGL和SNADGNGHGTHV,经序列比对确定该酶为新纤溶酶。体外实验显示,ACase可顺次降解人血纤维蛋白原α、β和γ链,具有明显的抗凝血作用。研究显示,茶树菇可以作为功能性食品基料用于开发降低血栓倾向的功能食品。In recent years,the increasing incidence of thrombotic diseases has seriously threatened human life and health.Some functional compounds in foods can increase the activity of human fibrinolytic system,thus gradually restore human's normal physiological state and reduce the tendency of thrombophilia.A novel fibrinolytic enzyme was purified from the fruiting body of Agrocybe aegerita by ammonium sulfate precipitation,Sephadex G-25 gel filtration,CM-Sepharose FF ion exchange,Source 15PHE hydrophobic chromatography and Mono S ion exchange.The fibrinolytic enzyme had a specific activity of 1731.05 U/mg,with the purification fold of 246.94.The enzyme was named ACase and comprised two subunits.The molecular weights of the two subunits were estimated to be approximately 31.4 kDa and 21.2 kDa,respectively,by SDS-PAGE.Only one band appeared on Native-PAGE and it was found to show fibrinolytic activity.The analysis by Edman degradation method showed that the N-terminal sequences of the two subunits were AIVTQTNAPWGL and SNADGNGHGTHV,respectively.The enzyme was identified as a new fibrinolytic enzyme by sequence comparison in GenBank.ACase could degrade the three chains(α,β and γ)of fibrinogen in order and exhibited the functionality of anticoagulant in vitro.Agrocybe aegerita has the potential application as functional food ingredient in food industry.

关 键 词：茶树菇子实体 纤溶酶 溶栓抗凝 功能性食品 分离纯化 

分 类 号：TS218[轻工技术与工程—粮食、油脂及植物蛋白工程]