纳豆枯草杆菌的筛选和纳豆激酶发酵条件优化  

Screening of Bacillus subtilis natto and optimization of nattokinase fermentation

作　　者：梅乐和[1] 胡升[1] 许静[2] 姚善泾[1] 郑晓冬[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]杭州中美华东制药有限公司,浙江杭州310011

出　　处：《浙江大学学报（工学版）》2004年第10期1355-1360,共6页Journal of Zhejiang University：Engineering Science

基　　金：国家自然科学基金资助项目(20176050);浙江省科技计划资助项目(2004C33036);浙江省回国人员基金资助项目;教育部回国人员基金资助项目;中德合作DAADCSC科技人员交流项目(PPP).

摘　　要：纳豆激酶是由纳豆枯草杆菌产生的一种具有纤溶活性的丝氨酸蛋白酶。针对目前常用菌株的产酶活力较低、不能完全满足要求的问题,采用酪蛋白平板初筛—摇瓶复筛的筛选策略,从纳豆中筛选获得了一株高产纳豆激酶的菌株,该菌株在筛选培养基上进行摇瓶培养,发酵液中纳豆激酶的酶活达到970IU/mL.用Plackett Burman方法对影响液体发酵的各因素的效应进行了评价,并筛选出了有显著效应的四个主要因素:胰蛋白胨浓度、种龄、发酵温度和Na2HPO4浓度;在此基础上,用最陡爬坡路径逼近最大产酶条件区域;最后通过中心组合实验及响应面分析优化了纳豆枯草杆菌液体发酵的条件.通过在初始发酵条件和优化发酵条件下的对比研究,液体发酵液中纳豆激酶浓度从1005IU/mL提高到1314IU/mL,表明响应面法优化可成功地用于纳豆枯草杆菌液体发酵的条件优化.Nattokinase, which is extracted from traditional Japanese food Natto is a fibrinolytic enzyme with strong and specific thrombolytic activity. A wild type Bacillus subtilis natto strain was screened from traditional Japanese food Natto with an improved medium-fibrinogen plate. The screening strategy included casein plate selecting and shaking culturing. The highest production of Nattokinase was 970 IU/mL. The fermentation condition of nattokinase production by Bacillus subtilis natto was optimized with response surface methodology (RSM). In the fist optimization step, Plackett-Burman design was used to evaluate the influence of related factors. Tryptone influenced nattokinase production positively while Na2HPO4, temperature and age/of inoculum influenced production negatively. The others had no significant influence on nattokinase production. The path of steepest ascent was used to approach the optimal region of the fermentation condition subsequently. The concentrations of tryptone and Na2HPO4, and the age of inoculum were further optimized using central composite designs and response surface analysis. The optimized condition allowed nattokinase production to increase from 1005 IU/mL to 1314 IU/mL.

关 键 词：纳豆激酶 纳豆枯草杆菌 Plackett Burman设计 响应面法 

分 类 号：TQ920.4[轻工技术与工程—发酵工程]