Cloning And Expression Of Class Ⅱa Bacteriocins Produced By Lactic Acid Bacteria

Bacteriocins produced by lactic acid bacteria (LAB) are a heterogeneous group of activated peptide or proteinous material, most of which are provided with inhibition for bacteria in near relation, and the minority with wider bacteriostatic spectra. Bacteriocins can be divided into four types. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their activities and potential applications. They have first attracted particular attention as listeria monocytogenes compounds and are now believed to be the next in line if more bacteriocins are to be approved in the future. Because of his antilisterial effectiveness, class IIa bacteriocins have significant potential as biopreservatives in a large number of foods. Each of lactic acid bacteria produces at least one or more of the bacteriacin. And so far only 29 class IIa of bacteriacins were found, most of which were discovered in the 1990s. Isolation and purification of the protein from native hosts purification are time-consuming, laborious processes, which usually result in low protein yield. So it is much difficult to find a new class IIa bacteriocin now. But we can continue to study the class IIa bacteriocins by bioinformatics and molecular biology meathod.Sakacin P, which is a class IIa bacteriocin, is very active against the food pathogen Listeria monocytogenes. Pediocin PA-1 is one of IIa bacteriocins which have been used as food preservative. In this study, the clone vectors (pGMT-sakP, pGMT-ped), expression vectors (pET28a-sakP, pET28a-ped) of Pediocin PA-1 and Sakacin P were constructed, then the target proteins were successfully expressed in E. coli BL21 (DE3) and their antibacterial activity were also obtained. And we also found that with the reduction of incubation temperature, the expression level of soluble protein increases dramatically. These results will be helpful for establishing a good method for new IIa bacteriocin screening.By bioinformatic and database searching, a segment of genes which has high sequence similarities with the mature peptide of class IIa bacteriocin was found out. We named it NB-C1, and its function is not identified yet. Firstly, we synthesized the gene sequence of NB-C1 artificially, and constructed the clone vector (pGMT-C1), expression vector (pET28a-C1). Then the target protein was successfully expressed in E. coli BL21 (DE3) and its antibacterial activity against listeria monocytogenes was also obtained. Therefore, we can conclude that NB-C1 is a new class IIa bacteriacin produced by LAB. The successful cloning and expression of biologically active sakacin P and NB-C1 in E. coli BL21 (DE3) may be helpful to determine more novel class IIa bacteriocins efficiently and conveniently from LAB genomes.