| Summary: | 碩士 === 元智大學 === 生物科技與工程研究所 === 104 === Nisin is one of class I lantibiotics that could be produce by Lactococcus lactis, containing one or more thioether bonds. Nisin is small sized in M.W(3.4 kDa), 34-amino acid, and has five characteristic lanthionine rings. It was widely used in food industry as a preservative, capable as starter-derived inhibitors to prevent growth of spoilage and pathogenic microorganisms, (eg., Gram-positive bacteria, anaerobe), also can extend the shelf life of food. However, the major limitation of nisin application is high cost of production due to low nisin productivity during fermentation of L. lactis. To achieve higher productivity and avoid lactic acid inhibition of L. lactis, constructing a promising nisin-production strain for large-scale nisin production would be of great importance. As a matter of fact, derivatives of Bacillus subtilis have been widely exploited for industrial production of biological materials. Bacillus subtilis is a gram-positive bacterium which owned high growth rates and was not capable to induce endotoxins. In this study, a nisin resistant recombinant B. subtilis was used as a host to be constructed to overcome the nisin sensitivity of B. subtilis with introduction of nisin resistance genes nisI or nisFEG from L. lactis. The findings demonstrated that both recombinant B. subtilis / pHY300PLK-nisI and B. subtilis / pHY300PLK-nisFEG owned the ability to sustain nisin concentration to 650 ppm in the culture. In addition, recombinant B. subtilis / pHT254-nisI , B. subtilis / pHT254-nisFEG and B. subtilis / pHT254-nisIFEG were found to sustain nisin concentration 100~1600 ppm. More important is after induced with 1 mM, 10 mM IPTG, recombinant strains increase nisin resistance significantly. Comparison revealed that recombinant B. subtilis / pHT254-nisI was the most appropriate strain of nisin resistance. Then, B. subtilis / pHT254-nisI was treated with UV radiation and chemical mutagens (100 μg / mL of Ethyl Methane Sulfonate; EMS). The results also showed that both of methods enabled recombinant B. subtilis / pHT254-nisI to sustain nisin concentration to 3000 ppm. This strain could sustain with 1600 ppm of nisin for fifteen times by repeated batch culture. This result shows that recombinant B.subtilis / pHT254-nisI could be a host on production of nisin for further applications.
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