Construction And Cultural Condition Optimization Of Recombinant Strains Of Nisin

Nisin,produced by Lactococcus lactis,is one of the antibiotic polypeptide.It can inhabit most of the gram positive pathogenic bacteria.Nisin is widely used in food industry for preservation.As a kind of Natural Food Preservative it is widely used in food preservatives.With the development of molecular biology,more and more scholars and researchers try to through genetic engineering methods produce nisin so as to improve its production.Although Lactococcus lactis is also a kind of bacteria the gram-positive pathogenic bacteria.But because of the resistance gene nisI and nisFEG,the Lactococcus lactis can defense the toxicity of itself.Primers were designed according to the gene sequence of nisI and the gene sequence was then amplified through PCR employing genomic DNA of Lactococcus ssp.lactis as template.After TA clone and double digestion then connecting operation towards the nisI and pMG36e vectors,the recombinant plasmids were then transformed to the E.coli JM109.Transformants with recombinant plasmids were screened and identifiedby PCR and double digestion of restriction enzyme,with the result that the target gene fragments were successfully inserted into the recombinant plasmids.The recombinant plasmid was introduced into the host stain Lactococcus lactis by electroporation.Named the successful engineering strains of lactic acid bacteria of LAB-pMG36e-nisI.The transcription level of nisI in both engineering strains and the parent strains were further detected by RT-PCR.We found that the nisI gene in engineering strains LAB-pMG36e-nisI had a higher expression level than the parent strains LAB in 6 h,9 h and 12 h.Fermentation medium and culture conditions were also investigated through single factor experiment and responding surface analysis.The optimal culture conditions were as follows:8 h for seed liquid culture time;7.5 for initial pH;3%for inoculation amount;The optimal constitution of culture medium was sucrose with a concentration of 26.70 g/L.peptone,yeast extract powder,beef extract,disodium hydrogen phosphate and magnesium sulfate with a concentration of 22.68 g/L,7.00 g/L,7.00 g/L,21.80 g/L and 0.20 g/L respectively,The production of nisin is from 1233.35 IU/mL to 2163.21 IU/mL.The recombinant strains and the parent strains were cultured With the same optimized medium and cultivation conditions we found that the production of nisin in the parent strains is 1918.35 IU/mL.The recombinant strains production of nisin is 12.76%higher than the parent strains.