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標題: 利用乳酸鏈球菌表現甜味蛋白質
Expression of the recombinant sweet protein brazzein by food grade Lactococcus lactis
作者: 翟爾雅
Chai, Erh-Ya
關鍵字: Lactococcus lactis;乳酸鏈球菌;Brazzein;甜味蛋白質
出版社: 食品暨應用生物科技學系所
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Lactococcus lactis被認為是異源蛋白質表現、分泌及遞送的優良宿主,具有長久發酵食品使用歷史,因此被認為是GRAS(generally recognized as safe)及安全菌株,作為異源蛋白質表現宿主具有應用於食品工業上之潛力。Brazzin為甜味蛋白質的一種,源自非洲果實Baillon中萃取出的水溶性蛋白質,與人類味蕾結合可產生甜味,其結構不易受pH值與溫度變化而影響且無後苦味,可用作天然低卡路里甜味劑。然其含量低,不易大量生產,使其在食品業應用方面受限,經濟效益低。本實驗構築MNICE與MPHI系統並將其應用於此重組甜味蛋白質之生產。

實驗中所有表現系統之培養基皆使用先前實驗室開發之FMB培養基,其成分來源安全且有助於提高菌體密度,所應用之蛋白質生產系統可視為食品級表現系統。實驗首先構築酸誘導系統,分為含有5UTL與不帶有此結構之表現系統,偵測重組甜味蛋白質表現發現不帶有此結構之MPHI系統可受酸誘導調控表現蛋白質,而帶有5UTL之MPHI系統因酸誘導調控之D box出現缺失,故只具有蛋白質表現能力而不具受乳酸調控之能力。接著表現系統使用PnisA啟動子帶有5UTL搭配先前實驗室挑選之最佳訊息胜肽SPsacB 分泌表現重組甜味蛋白質,其在蛋白質產量方面明顯優於MPHI系統。實驗在MNICE系統中找到蛋白質產量最高時間在誘導後12小時,而nisin誘導最佳劑量為60 μg/ml,可於誘導後4小時得蛋白質產量260.9 μg/ml。甜味測試方面由於發酵上清液之味道受FMB培養基之干擾,雖可以50 ~ 300 mg/ml cysteine作用37℃/1 hr增進甜味,但仍無法確切比較出甜度差異性。

The lactic acid bacterium Lactococcus lactis is considered as an advan- tageous host for protein expression, secretion and delivery. The bacterium has an established safety profile through its long use in fermented dairy products and is considered as a GRAS(generally recognize as safe)microorganism. Brazzein is a sweet protein which was found in a tropical plant. The brazzein is impractical due to its low content in nature fruit and thus limiting its availability in food industry. In this study, we construct MNICE and MPHI induction expression system to produce sweet protein by L. lactis.

All the expression systems used the FMB medium, which was a component sources safe and achieved high cell density, and thus considered as food grade expression system. In the first section, we examined the pH-inducible system, using P170 promoter with(MPHI) or without slpA 5UTL(PHI) to express rbrazzein protein. Results showed that PHI system exhibited pH-inducible and secretion ability in L. lactis NZ9000. MPHI system could not be induced by lactic acid, and was found a deletion on promoter D box. In another section, our lactococcal expression/secretion system, using both PnisA(with 5UTL), an expression cassette tightly controlled by environmental nisin(MNICE), and a consensus signal peptide, SPsacB, allows efficient production and secretion of the rbrazzein, as compared by protein yields (protein amount/biomass) to MPHI expression systems. In MNICE system, results showed the nisin induction time 12hr achieved highest rbrazzein expression and the best induction concentration was 60 μg/ml nisin which achieved rbrazzein protein 260.9 μg/ml at induction time 4 hr. The taste experiment was interfered by the heavy flavor of medium, adding 50 ~ 300 mg/ml cysteines and acting 37℃/1 hr could promote the sweetness, but still could not quantitative analysis the sweetness.
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