Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90222
標題: 不同訊息胜肽在枯草桿菌中分泌重組第一型抗凍蛋白類似物之最適化及其功能性測試
The optimum production of recombinant type I antifreeze protein analogue and functional analysis with various signal peptides by Bacillus subtilis
作者: 戴嘉言
Jia-Yan Dai
關鍵字: Recombinant type I AFP (rAFP);Bacillus subtilis;signal peptide;single plasmid induction system;constitutive system;dual plasmid induction system;重組第一型抗凍蛋白;枯草桿菌;訊息胜肽;單質體誘導系統;持續型系統;雙質體誘導系統
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摘要: 
Antifreeze proteins (AFPs) play an important role in the freezing tolerance of various organisms such as fish, plants, fungi 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 are gram-positive、facultative anaerobe and endospore -forming bacteria. They has been classified as GRAS (generally recognized as safe) microorganism by FDA(Food and Drug Administration) 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, single plasmid induction system of B. subtilis were constructed to express the rAFP . Constitutive and dual plasmid induction system with different signal peptides were constructed to observe the production yield,the results indicated that signal peptide sacB can help rAFP secretion,the others signal peptide is not the case.Optimize the rAFP production yield of B. subtilis (pSECS-7sacB-AFP) had the maximum yield of 664.63 mg/L.

The application of rAFP in snow ice showed that rAFP together with other materials such as milk、sugar and corn starch improved the Preference of
density and overall.

抗凍蛋白(antifreeze proteins , AFPs)為某些生存於極地或寒冷地區之生物,如魚類、植物、真菌及昆蟲體內所特有的蛋白質,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組織、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)為革蘭氏陽性(gram-positive)、兼性厭氧(facultative anaerobe)之產孢桿菌,且經美國食品暨藥物管理局(Food and Drug Administration, FDA)認可為GRAS (generally recognized as safe)級之宿主,常被運用於發酵工業。而在遺傳工程研究中,常被使用做為宿主細胞以表現異源性蛋白質。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。

本實驗成功建立了枯草桿菌單質體誘導型表現系統,表現重組第一型抗凍蛋白質類似物。另外,也成功將持續型及雙質體誘導型系統進行訊息胜肽的置換,其中以訊息胜肽sacB可大量幫助成熟蛋白質分泌至胞外,而於其他訊息胜肽中,可於胞外發現帶有大量訊息胜肽未剪切之目標蛋白。其中B. subtilis WB800 (pSECS-7sacB-AFP)轉型株,經小型發酵可得到最高蛋白產量為664.63 mg/L。將上清液純化所得到之重組蛋白質應用於雪綿冰品評中,結果顯示添加抗凍蛋白質的雪綿冰組別在整體喜好度上領先其他組別,具有較好的口感。
URI: http://hdl.handle.net/11455/90222
Rights: 同意授權瀏覽/列印電子全文服務,2017-08-06起公開。
Appears in Collections:食品暨應用生物科技學系

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