Cloning of nis gene and Nisin purification from Lactococcus lactis subsp. lactis Fc2

Abstract

The present study was focused on nisin produced by Lactococcus lactis subsp. lactis (Fc2 isolate).  The isolate was identified by 16S rRNA using specific universal primer. The obtained cell-free supernatant inhibits the growth of Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis by using agar diffusion assay. Nisin structural gene was detected by polymerase chain reaction with nisin gene-specific primer followed by cloning in PGEM-T easy vector, transformation in E. coli JM109 and direct sequencing of the construct. The Nis gene which encoded nisin Z showed identical sequences to Nis Z in Genbank with accession number AB727286, as indicated by the substitution of asparagine residue instead of histidine at position 27. In this way, it was established that the nis Z gene for nisin Z production is widely distributed. The purified nisin by chloroform extraction was analyzed on 20% SDS-PAGE and gave sharp band at ~ 3.4 kDa. The 3 dimension structure of the purified Nisin was studied by CPHModels as pdb with chimera program.

Keywords: Lactococcus lactis, nis cloning, 16S rRNA, chloroform extraction and SDS-PAGE

African Journal of Biotechnology, Vol 13(53) 4711-4719