Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52018
標題: The optimum production of recombinant type I antifreeze protein analogue by Bacillus subtilis and application on probiotic and cell cryopreservation
枯草桿菌生產重組第一型抗凍蛋白質類似物之最佳化及於益生菌與細胞冷凍保存應用
作者: 詹雅婷
Chan, Ya-Ting
關鍵字: Recombinant type I AFP (rAFP);重組第一型抗凍蛋白質類似物;Bacillus subtilis;cryopreservation;C2BBel cell;Lactobacillus;枯草桿菌;冷凍保護;細胞;益生菌
出版社: 食品暨應用生物科技學系所
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摘要: 
抗凍蛋白質(antifreeze proteins, AFP)存在於某些寒帶地區生物如魚類、植物和細菌體內,具有修飾冰晶構形,抑制冰晶生長及抑制冰晶再結晶之功能。可應用於冷凍食品之品質保存或細胞、精卵之冷凍保存及非耐寒生物轉殖抗凍蛋白基因降低寒害等。
枯草桿菌(Bacillus subtilis)為GRAS (generally recognized as safe)級之菌種,常被運用於發酵工業。此外在遺傳工程研究範圍中,亦常被使用做為宿主細胞表現異源蛋白質。本實驗室先前已成功建立枯草桿菌分泌表現重組第一型抗凍蛋白質類似物之系統,但產量仍有待提升。
本研究建立枯草桿菌持續型和誘導型表現系統表現重組第一型抗凍蛋白質類似物,其中以B. subtilis(pSECS-6AFPT) 發酵可得最高產量為872 mg/L。將上清液純化所得之重組第一型抗凍蛋白質類似物經加熱1.5小時或改變緩衝液pH值範圍於2~12後仍具有抗凍活性。重組第一型抗凍蛋白類似物應用方面,益生菌冷凍保護部分,重組第一型抗凍蛋白類似物搭配海藻糖,脫脂奶粉等保護劑可提高Lactobacillus paracasei菌體的冷凍儲存存活率。於菌體凍乾實驗中,在適當儲藏條件下,重組第一型抗凍蛋白類似物可略為提升L. paracasei存活率。細胞冷凍保護部分,重組第一型抗凍蛋白類似物雖無顯著差異但可略為提升DMSO保護劑處理之C2BBel細胞冷凍存活率。本實驗室研發之重組第一型抗凍蛋白質未來可嘗試應用於其他生物素材之冷凍保護,增加其應用潛力。

Antifreeze proteins (AFPs) play an important role in the freezing tolerance of various organisms such as fish, plants, and bacteria who live in supercooling conditions. It modifies the shape of ice crystal, inhibits ice growth, and represses the ice crystals from recrystallization. AFPs have potential to apply in food technology, cryopreservation, cryosurgery and transgenic technologies.
Bacillus subtilis has been classified as GRAS (generally recognized as safe) microorganism by FDA and considered as an excellent host for the secretory production of heterologous proteins.In our previous study, Synthetic gene encoding the recombinant type I AFP (rAFP) analogue has been expressed in Bacillus subtilis, but the production yield needed further improvement.
In this study, constitutive and inducible system of B. subtilis were constructed to optimize the rAFP production yield. B. subtilis (pSECS-6AFPT) had the maximum yield of 872 mg/L. The rAFP heated for 1.5 hr or changed in various pH buffers ranging from 2-12 still retained their antifreeze activity.
The application of rAFP in probiotic cryopreservation showed that rAFP together with other cryoprotectants such as trehalose or skim milk improved the viability of frozen Lactobacillus paracasei. In proper storage condition,rAFP slightly improved the viability of lyophilized L. paracasei. In C2BBel cell cryopreservation experiments, rAFP slightly but not significantly improve the DMSO treated frozen C2BBel cells viability. The potent application of rAFP on biological materials cryopreservation is highly expected in the future.
URI: http://hdl.handle.net/11455/52018
其他識別: U0005-2607201116174000
Appears in Collections:食品暨應用生物科技學系

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