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Analysis of sugar moiety of enterocin from Enterococcus faecium N15

屎肠球菌 N15 肠菌素糖部分的分析

基本信息

批准号：
12650785
负责人：
SHIOYA Suteaki
金额：
$ 1.98万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12650785/
关键词：
EnterocinA Bacteriocin Shuttle vector Enterococcus faecium Structure of sugar moiety E.faecium

项目摘要

Some lactic acid bacteria produce antimicrobial substances such as bacteriocin which could preserve food. We isolated Enterococcus faecium N15 producing the antimicrobial substance named enterocin from rice bran. The activity of this enterocin is lost with the alpha-amylase treatment, implying that this has a sugar moiety which is important for the activity. We also have shown that E. faecium N15 has a entA gene as previously reported. In this study, we expressed the entA gene in E. faecium N15 and analyze the gene product.Polymerase chain reaction was performed to amplify the fragment including entA gene and its upstream region. The resultant fragment was inserted into the E. coli-lactic acid bacteria shuttle vector to construct the expression vector of entA. The electroporation was performed to insert the vector into E. faecium N15. A transformant was used for investigation of expression of entA. However it failed. Therefore, we tried to express the entA gene under P32 promoter which has been known to promote strongly the expression of a gene in lactic acid bacteria. So we constructed a new expression vector containing P32 promoter and performed the electroporation. On the other hand, entA gene was expressed using the E. coli expression system in order to characterize the entA gene product. The resultant protein was shown to be resistant to heat and pH change. This properties are same as those of roughly purified enterocin from culture of E. faecium N15. However, the result was different in alphaamylase sensitive. This result suggested that entA gene product and the roughly purified enterocin from a culture of E, faecium N15 are different proteins.

一些乳酸菌产生抗菌物质，如细菌素，可以保存食物。从米糠中分离出粪肠球菌N15，产生抗菌物质肠球菌素。这种肠球菌蛋白的活性随着α -淀粉酶的处理而丧失，这意味着它含有对活性很重要的糖部分。我们还发现，如之前报道的那样，粪肠杆菌N15有一个entA基因。在本研究中，我们在粪肠杆菌N15中表达了entA基因并分析了基因产物。采用聚合酶链反应扩增包含entA基因及其上游区域的片段。将所得片段插入大肠杆菌-乳酸菌穿梭载体中，构建entA表达载体。采用电穿孔法将载体插入粪肠杆菌N15中。利用转化法研究entA的表达情况。然而它失败了。因此，我们尝试在P32启动子下表达entA基因，P32启动子在乳酸菌中可以强烈促进一个基因的表达。因此，我们构建了一个包含P32启动子的新表达载体，并进行了电穿孔。另一方面，利用大肠杆菌表达系统表达entA基因，以表征entA基因产物。结果表明，所得蛋白质对热和pH值变化具有抗性。这种特性与从粪肠杆菌N15培养物中大致纯化的肠球菌素相同。但对α淀粉酶敏感的结果不同。这表明entA基因产物与从E， faecium N15培养物中大致纯化的肠蛋白是不同的蛋白。