| Summary: | 碩士 === 中興大學 === 食品暨應用生物科技學系 === 95 === The researches of the lactic acid bacteria (LAB) receive much concern in recent years. Among the published literatures, the genetically modified LAB and its application are attractive. Lactococcus lactis, a generally recognized as safe (GRAS) bacterium, is the most extensively studied LAB. It is widely used for industrial fermentation and has recently become an attractive host for the production of heterologus proteins. In this study, we used L. lactis NZ9000 as host, aimed to set up a food grade expression-secretion system to produce recombinant antifreeze protein (AFP) analogue. AFPs, also known as thermal hysteresis proteins (THPs), were found in a wide range of organisms living in cold ambient conditions. The ability of AFPs to influence ice crystal growth has led these proteins used potentiality in a wide variety of applications, such as use of them as food additives to enhance the quality and shelf-life of frozen foods. In previous studies, a synthetic gene encoding the recombinant type I AFP (rAFP) analogue has been expressed in Escherichia coli, Bacillus subtilis and L. lactis in our labortory. In this study, we have investigated the influence of various signal peptides, transcription terminators, and media on the secretory production of rAFP in L. lactis.
In the first section, we examined the effect of the signal peptides from L. lactis secreted protein (Usp45), B. subtilis levansucrase (SacB), B. subtilis YaB alkaline elastase subtilisin (Ale) and Lactobacillus acidophilus S-layer protein (SlpA) in secretion yield of rAFP. Results showed that SPsacB exhibited the best secretion level in L. lactis NZ9000. When AE was fused immediately after the signal peptide cleavage site, secretion yield of rAFP was increased more than fused to LEISSTCDA. By the results, we choose SPsacB and fused AE after cleavage site as the secretion signal to produce rAFP. And then, we replaced the pQE terminator with slpA terminator to achieve the food grade production system. However, the entire accurate TerslpA was unable to obtain and the point mutation in TerslpA were happened during the construction. Therefore, three TerslpA which mutated in different base pair were subject to compare. Result showed that mutated in the 24th position of TerslpA exhibited the best secretion yield in expression of rAFP. A biosafety culture medium was developed to express the rAFP with best secretion and terminator signal at high level in L. lactis.
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