Analysis of sugar moiety of enterocin from Enterococcus faecium N15

一些乳酸菌产生细菌素等抗菌物质，可以保存食物。我们从米糠中分离到一株产抗菌物质肠球菌素的粪肠球菌N15。这种肠球蛋白的活性随着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。我们还发现粪肠球菌N15具有先前报道的entA基因。在本研究中，我们在粪肠球菌N15中表达了EntA基因，并对其产物进行了分析，通过聚合酶链式反应扩增了含有EntA基因及其上游的片段。将得到的片段插入到大肠杆菌-乳酸菌穿梭载体中，构建EntA的表达载体。电穿孔将载体插入到粪肠球菌N15中。利用转化子对EntA的表达进行研究。然而，它失败了。因此，我们尝试在P32启动子的作用下表达Enta基因，该基因在乳酸菌中具有强烈的促进基因表达的作用。因此，我们构建了含有P32启动子的新表达载体，并进行了电穿孔实验。另一方面，利用大肠杆菌表达系统对EntA基因进行了表达，并对EntA基因产物进行了鉴定。结果表明，所合成的蛋白对高温和pH变化具有抵抗力。这些性质与从粪肠球菌N15培养中粗提纯的肠球蛋白相同。但对α-淀粉酶敏感的结果有所不同。这一结果表明ENTA基因产物与粪肠球菌N15培养物粗提纯的肠球蛋白是不同的蛋白质。

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.