豆豉纤溶酶的分离纯化及部分酶学性质研究

 目的 从豆豉中分离出具有强纤溶活性的菌株，从该菌株的发酵液中纯化出纤溶酶并对此纤溶酶的部分酶学性质进行研究。方法 利用硫酸铵盐析、DEAE-Sepharose Fast Flow阴离子交换色谱和Sephadex G-75凝胶过滤色谱等方法进行纯化；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。结果 从发酵液中获得了纤溶酶单一组分，表观相对分子质量为30×10³。该纤溶酶在50 ℃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。结论 获得了纤溶酶单一组分，为纤溶酶发酵产品的大规模纯化和进一步研制开发新的溶栓药物提供重要理论依据。

Purification and Characterization of the Fibrinolytic Enzyme in Fermented Soya Beans

OBJECTIVE To isolate bacterial strains with strongly fibrinolytic enzyme activity from Douchi and to purify and characterize fibrinolytic enzyme produced by the isolated strain.METHODS Fibrinolytic enzyme was purified by ammonium sulfate fractional precipitation， anion-exchange chromatography and gel filtration and identified by N-terminal amino acid sequence determination and MALDI-TOF-MS. RESULTS A single protein fraction with fibrinolytic activity was obtained from the liquid medium.The relative molecular weight of purified protein was 30×10³. 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 8.6， respectively.The result of N-terminal amino acid sequence determination and MALDI-TOF-MS showed homologies of up to 100% between the fibrinolytic enzyme and nattokinase. CONCLUSION A single fibrinolytic enzyme was purified， which provided the basis for large-scale production of fibrinolytic enzyme and might improve its potential application in thrombolytic therapy.