Analysis of sugar moiety of enterocin from Enterococcus faecium N15

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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.