纳豆中纳豆枯草杆菌的筛选和纳豆激酶的分离过程研究  

Screening of Bacillus subtilis natto from Traditional Japanese Food Natto and Separation of Nattokinase

作　　者：黄俊[1] 梅乐和[1] 胡升[1] 盛清[2] 许静 吴晖 姚善泾[1] 

机构地区：[1]浙江大学化学工程与生物工程系,浙江杭州310027 [2]浙江理工大学生命科学学院,浙江杭州310018 [3]中美华东制药有限公司,浙江杭州310011

出　　处：《高校化学工程学报》2005年第4期518-522,共5页Journal of Chemical Engineering of Chinese Universities

基　　金：国家自然科学基金(20176050);浙江省科技项目;教育部回国人员基金;浙江省回国人员基金

摘　　要：采用酪蛋白平板初筛-摇瓶复筛的筛选策略,从日本传统食品纳豆中筛选获得了一株高产纳豆激酶的纳豆枯草菌菌株,与国内公开报道的不同菌株相比,有更好的产酶能力。菌株经过液体发酵后获得含酶发酵液,采用硫酸铵盐析及SephadexG-75凝胶层析、SepharoseCMFastFlow离子交换层析对纳豆激酶进行了分离纯化,两步层析的纯化倍数和酶活回收率分别达到4.75、74.3%和1.32、77%,获得了纯度很高的纳豆激酶。Nattokinase has been found to be a strong fibrinolytic enzyme. A strain of Bacillus subtilis natto was screened from traditional Japanese food natto with an improved method. The screening strategy included the first selection with the casein plate and the further approach with shaking culture. Compared with the reports in China, the strain obtained in present work has the highest nattokinase productive ability and the highest production of nattokinase is 970 urokinase units per milliliter broth. After clarifying the broth, nattokinase was separated and purified with a series of suitable procedure including salt-out with ammonium sulfate, gel filtration with Sephadex G-75 and ion-exchange chromatography with CM Sepharose Fast Flow. The 20%-60% saturation of (NH4)2SO4 is suitable for the salt-out of nattokinase from fermentation broth, and the recovery of natlakinase is more than 90%. The purification factor of gel filtration and ion-exchange chromatography are 4.75 and 1.32, respectively, while the recovery are 74.3% and 77%, respectively. The specific activity of nattokinase purified is 16352 IU &middot mg-1, and the product shows only one stainable subunit on the gel of SDS-PAGE.

关 键 词：纳豆 纳豆激酶 纳豆枯草杆菌 分离纯化 

分 类 号：TQ920.4[轻工技术与工程—发酵工程] Q814.1[生物学—生物工程]