摘要
以日本纳豆、重庆江津豆豉、忠县豆腐乳为原料,采用酪蛋白平板初筛、摇瓶复筛,得到18株产溶栓酶的菌株.通过纤维蛋白平板实验,从忠县豆腐乳中筛选出1株具有较高溶栓活性的菌株FR-033(登录号:HQ009804),菌株经液体发酵后获得含酶发酵液,采用硫酸铵分段盐析及sephadexG-50凝胶层析进行纯化,获得较高纯度的溶栓酶,酶活力为2 163 IU/mL.进一步对FR-033菌株的形态和生理生化特征进行研究.利用细菌16S rDNA通用引物对16S rRNA进行PCR扩增并测序,得到1444 bp长度的DNA序列,通过BLAST软件在NCBI网站收索其同源性性序列,利用PAUP4.0和MAGE4软件绘制系统发育树,结果表明:菌株FR-033与枯草芽孢杆菌(Bacil-lus subtilis)的16S rRNA序列同源性达99%以上,在系统发育树中,菌株FR-033与枯草芽孢杆菌在同一支上,且遗传距离最短;FR-033的形态及大部分生理生化特征与枯草芽孢杆菌极为相似,表明该菌株是枯草芽孢杆菌(Ba-cillus subtilis)的一个高效的产溶栓酶菌株.
Bacillus natto can be separated from Japanese traditional fermented food and it has the function of dissolving thrombus.Whether similar strains can be separated and identified from Chinese traditional fermented foods needs further studies.In an experiment reported in this paper,imported Japanese natto,Jiangjin douche（a fermented soybean） and Zhongxian pickle bean curd were used as the raw materials.A total of 18 fibrinolytic enzyme-producing strains were obtained,using the method of casein flat as the initial screening and flask shaking as the second screening.In a subsequent fibrin plate experiment,a strain（FR-033）,which had high efficiency in dissolving thrombosis,was isolated from pickle bean curd（accession number： HQ009804）.The strain was treated with liquid fermentation,and a fibrinolytic enzyme-containing fermented liquid was obtained.The enzyme was purified with ammonium sulfate subsection salting-out and gel chromatography of sephadexG-50,and a fibrinolytic enzyme of high purity was obtained,with an enzyme activity comparable to that of urokinase（2 163 IU/mL）.A 1 444 bp fragment of 16S ribosomal RNA was obtained from total DNA of FR-033 by PCR amplification,using the universal primer of bacterial 16S ribosome DNA.Sequence analysis was carried out by Blast on NCBI website and a phylogenetic tree was constructed by PAUP4.0 and MAGE4 software.The results showed that FR-033（HQ009804） shared 99% 16S ribosomal RNA sequence homology with Bacillus subtilis.Strain FR-033 and B.subtilis were found to be on the same branch of the phylogenetic tree and their genetic distance was the shortest.Combining the similarity of morphological and physiological characters,FR-033 was identified as a highly efficient fibrinolytic enzyme-producing strain of B.subtilis.
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第6期85-93,共9页
Journal of Southwest University(Natural Science Edition)
基金
重庆市自然科学基金重点项目(No.CSTC2008BA5030)
