Construction And Application Of Gene Engineering Strain Of Nisin

Nisin,produced by Lactococcus lactis,is one of the antibiotic polypeptide.Nisin can inhabit most of the gram positive pathogenic bacteria,especially some bacteria and spore that can cause food corruption.Therefore it can be used for meat,wine,dairy preservatives.Becanse nisin is pure and natural preservative,produced by Lactococcus lactis fermentation,which can be hydrolyzed into a variety of amino acids by enzymes in the body.It has no residue and resistance,do not inhabit human probiotics.It has a huge market efficiency and economic value.Because of the low production level,nisin's price is always higher than other preservative,which will hinder nisin's generalization.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.NisI and ABC transporter which are encoded by the resistance gene nisI and nisFEG can defense the toxicity of itself.Structural gene nisA is required for pre-nisin synthesis.Throughmodification,leader peptide biosynthesis,Pre-nisin come to mature nisin.Primers were designed according to the gene sequence of nisA,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 nisA and pMG36 e vectors.After double digestion then connecting operation towards the nisI and pMG36e-nis A vectors,the recombinant plasmids were then transformed to the E.coli JM109.Transformants with recombinant plasmids were screened and identified by 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.Take the production of nisin as indexes to screen out transformant.Then we found a high-yielding gene engineering strain,AI-5.By measuring the cells growth curves,resistance capabilities to nisin and genetic stability,we grasped the growth rule of AI-5.At last,AI-5 was confirmed as a stable high yield strain for nisin.Fermentation medium and culture conditions were also investigated through single factor experiment,orthogonal experimental design.The optimal culture conditions were as follows: 30? anaerobic fermentationfor 16 h.Use 5M NaOH to adjust fermentation broth pH every two hours to 6.5 from 6 h to the end.The optimal constitution of culture medium was sucrose with a concentration of 30 g/L;peptone,yeast extract powder,beef extract,disodium hydrogen phosphate,magnesium sulfate with a concentration of 25 g/L,7.00 g/L,7.00 g/L,20 g/L and 0.3 g/L respectively.Gene engineering strain's production of nisin reached 2891IU/mL,4 times of the production obtained in pre-optimized medium and culture conditions.Production increased 20.07% compared to the wild-type strain in the same cultivation conditions and medium.