Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52150
標題: 以地衣芽孢桿菌表現和純化重組甜味蛋白質之最適化及其功能特性之探討
Optimization of recombinant sweet protein brazzein expression, purification and functional analysis by Bacillus licheniformis
作者: 洪千雅
Hung, Chien-Ya
關鍵字: 重組甜味蛋白質;recombinant sweet protein rbrazzein;地衣芽胞桿菌;分泌生產;Bacillus licheniformis;secretion
出版社: 食品暨應用生物科技學系所
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摘要: 
甜味劑在人的日常飲食中扮演著很重要的角色,然而過度攝食卻造成健康風險。近年來取代傳統甜味劑的低熱量甜味劑需求越來越大。甜味蛋白質具有潛力能夠做為低熱量甜味劑而應用於食品、飲料和醫藥上。Brazzein為目前已知分子量最小的甜味蛋白質,它的甜度高、風味與蔗糖相似,而且pH值和熱穩定性佳,具有潛力能應用於加工食品上。
枯草桿菌(Bacillos subtilis)與地衣芽孢桿菌(Bacillus licheniformis)皆為革蘭氏陽性 (gram-positive)之產孢桿菌,並且為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,具有分泌蛋白質之能力,於遺傳工程上能夠做為異源性蛋白質表現之宿主。
本研究以枯草桿菌和地衣芽胞桿菌作為表現重組甜味蛋白質之宿主,藉由構築不同的訊息胜肽質體,觀察枯草桿菌和地衣芽胞桿菌於不同蛋白質分泌途徑中表現重組甜味蛋白質的情形,從結果發現枯草桿菌皆無法表現重組甜味蛋白質到胞外,只有於胞內有偵測到帶有訊息胜肽的重組甜味蛋白質。而地衣芽胞桿菌雙質體誘導系統能夠成功於胞外表現重組甜味蛋白質。由兩公升地衣芽胞桿菌36小時發酵液純化重組甜味蛋白質,經由濃縮透析可以得到約10 mg之產量,而且純度可達85%。將純化重組甜味蛋白質進行蛋白質身分鑑定,確定為brazzein,而且結構中有正確形成雙硫鍵。經感官品評分析其甜度為同質量濃度蔗糖的266倍,每分子的甜度為蔗糖的4957倍,而且於pH 5~9之間具有良好的熱穩定性。由此可證實地衣芽胞桿菌蛋白質表現系統能夠生產具活性之重組甜味蛋白質,有應用於食品工業上之潛力。

The sweetener plays an important role in our daily diet. However, the excessive intake of carbohydrates lead to health risk. In recent years, the search for low-calorie sweeteners to substitute sugar is highly demand. The sweet-tasting protein has potential as low-calorie sweeteners that can be used to replace sugars in food, beverage, and medicines. Among them, brazzein is an attractive candicate sweetener because of its small size(53 amino acid residues), high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges.
Bacillus subtilis and Bacillus licheniformis are gram positive and endospore-forming bacteria. They have been regarded as GRAS (generally recognized as safe) by FDA (Food and Drug Administration). They have the ability to secrete protein and can be used as good hosts for heterologous protein expression.
In this study, B. subtilis and B. licheniformis are used as hosts to express the recombinant sweet protein rbrazzein. Plasmids with different signal peptides were constructed to observe the secretion of rbrazzein by different protein secretion pahway. The results indicated that rbrazzein couldn''t be secreted by B. subtilis. However, the rbrazzein is successfully expressed by B. licheniformis dual plasmid system. The optimized rbrazzein purification achieved approximately 10 mg purified rbrazzein from 36-hour fermented B. licheniformis culture.The rbrazzein was examined to confirm the disulfide bond existence. The purified rbrazzein was 266 times sweeter than sucrose on a weight basis, and about 4957 times sweeter than sucrose on a molar basis. It is also with good heat stability between pH 5 to pH 9. These results demonstrated that B. licheniformis expression system is useful to produce active recombinant sweet protein brazzein and can be useful as potent sweetener producing system and applied in food industry
URI: http://hdl.handle.net/11455/52150
其他識別: U0005-2607201312470600
Appears in Collections:食品暨應用生物科技學系

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