豆豉纤溶酶的分离纯化酶学性质的研究及微胶囊的制备微生物学专业论文.docxVIP

摘要摘要 摘要 摘要 豆豉纤溶酶(E。C。3。4．21．7)是一种从中国传统食品豆豉中分离出来的具体较高纤溶活性的 丝氨酸蛋白酶。它不仅能直接溶解纤维蛋白，抑制血栓形成，并且能够抑制机体凝血，抗血 小板聚集等功能。 本论文对DC．10产纤溶酶的分离纯化、酶学性质、微胶囊的制备及性质进行了系统的 研究。 (1)采用TAME法作为豆豉纤溶酶的测定方法，操作简单、快速；精密度较高，重现性 高。 (2)对DC．10菌株产纤溶酶的分离纯化进行了研究。利用硫酸铵盐析、DEAE-Sepharose Fast Flow阴离子交换层析和Sephadcx G．75凝胶过滤层析等方法进行纯化，获得了纤溶酶单 一组分，表观分子量为30 kDa，最终纯化倍数达lO．17，回收率为16．69％。 (3)对DC．10产纤溶酶的部分酶学性质进行了研究并利用N．末端氨基酸序列测定及基 质辅助激光解吸电离飞行时间质谱(MALDI-TOF．MS)对此酶进行鉴定。该纤溶酶在50。C以 下和pH 7．0-9．0之间保持稳定，最适温度为35℃；最适PH为8．6；N-末端氨基酸序列及飞 行时间质谱测定结果都表明该酶与纳豆激酶氨基酸序列有着极高的相似性。 (4)通过挤压法豆豉纤溶酶微胶囊，以海藻酸钠一氯化钙一壳聚糖体系制备出微胶囊。 检测结果表明，微胶囊酶活可达到162．96 IU／mL，包埋率为90．25％。经胃酸、胆汁酸处理 后酶活1 14．07 IU／mL以上，对酸有很高的耐受力；经人工肠液处理35 min，微胶囊几乎全 部崩解，肠溶释放率可达到97．42％。 本课题对新型溶栓药物—一豆豉纤溶酶的分离纯化、酶学性质、微胶囊的制备进行了系 统研究，目前还没有见到关于豆豉纤溶酶微胶囊的报道。本课题的研究结果为豆豉纤溶酶发 酵产品的大规模纯化和迸一步研制开发新的溶栓药物剂型提供重要理论依据。 关键词纤溶酶；豆豉：分离纯化；性质；微胶囊 东北林qk大学硕上．学位论文Abstract 东北林qk大学硕上．学位论文 Abstract Dochi Fibrinolytic Enzyme正．C．3．4．2 1．7)is a kind of serine protease and acts as觚importam fibrinolytic and antithrombotic enzyme． Doc“Fibrinolytic Enzyme WaS purified and the characteristics of Dochi Fibrinolytic Enzyme were meaSured．A novel methed WaS developed for the preparation of mirocapsule of the Fibrinolytic Enzyme． (1)The TAME method WaS applied to determinate the activity of Fibrinolytie Enzyme，which provided a rapid means ofdetection． (2)Fibrinolytic enzyme was purified by ammonium sulfate fractional precipitation,anion- exchange chromatography and gel filtration．A single protein fl删on with fibrinolytic activity WaS obtained from the liquid medium．．nle relative molecular weight of purified protein is 30 kDa，The purification fold WaS 10．17 and recovery oftotal laccase activity w舔16．6钟么 (3)The purified fibrinolytic enzyme was stable blow 50℃and maintained its activity at pH 7．0-9．0．The optimal temperature and pH were 35℃and 9．0，respectively．We searched the Database for the ressult of MALDI-TOF—MS．Homologies of up to 100％were found with the fibrinolytic enzyme and nattokinase． (4)We research the extrusion method of miero