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標題: 以染色體嵌入技術製備枯草桿菌 γ-PGA 生產株 及其發酵生產
Construction of γ-PGA-producing strain by chromosomal integration in Bacillus subtilis and optimization of fermentative production
作者: 鄭浚鳴
Jun-Ming Zheng
關鍵字: γ-PGA, CTAB;fermentation broth;quantitative analysis;chromosome integration;Bacillus subtilis;Plackett-Burman design;optimized medium;γ-PGA;溴化十六烷基三甲銨(CTAB);發酵液;定量分析;染色體嵌入;枯草桿菌;Plackett-Burman 因子設計;優化培養基
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Poly-γ-glutamic acid (γ-PGA) is a versatile high molecular biopolymer material, which has been applied in food, medical, cosmetic, animal feed,and wastewater industry. Enhanced production of γ-PGA is highly recommended.
In order to enhance the screening of high γ-PGA yielding strain and quantitation of γ-PGA. A rapid quantification of γ-PGA was established in the beginning of this study. CTAB binds specifically to γ-PGA and form a water-insoluble, highly dispersed micelle-like complex, resulting in an increase in turbidity. The turbidity-based calibration curve of γ-PGA was
established as y = 0.0055x – 0.0349 (x and y represent the concentration of γ-PGA and the mixtures turbidity at 400 nm) with a good linearity. The turbidimetric method has advantages of convenience , simplicity and good repeatability and can be used for γ-PGA concentration detecting in the fermentation broth.
The host Bacillus subtilis WB800, which possesses the γ-PGA synthesizing genes, pgsBCAE, on its chromosome cannot produce γ-PGA. The efficient constitutive or inducible synthetic expression control sequence (SECS) was introduced into the upstream region of the pgsBCAE genes, resulted in γ-PGA-producing B. subtilis transformants. The transformant strain B. subtilis Dc8006 stably produced high levels of γ-PGA in medium A without extra glutamate supplement.
To evaluate the effect of different culture parameters on production of γ-PGA, Plackett–Burman factorial design was preceded. Twelve varients were examined for their significance on γ-PGA production. Based on
statistical pre-optimized medium analysis, optimized medium PBD were subjected for fermentation, and achieved 35.2 g/l γ-PGA yield, which is 1.5 times than the original medium A.

γ-PGA 是一種高分子的生物材料,已被運用於食品、醫藥、化妝品、飼料及廢水處理工業上,提高 γ-PGA 的生產有其必要性。
為了提升篩選 γ-PGA 高產轉形株及定量 γ-PGA 之便利性,本研究首先建立一個 γ-PGA 快速定量法,利用 γ-PGA 與溴化十六烷基三甲銨
曲線。標準曲線公式為 y = 0.0055x – 0.0349( x 和 y 表示混合物在 400nm波長下 γ-PGA 的濃度及濁度) ,具有良好的線性。應用比濁法測定 γ-PGA的含量具有快速、簡潔、重現性好等優點,可用於發酵液中 γ-PGA 濃度的檢測。
宿主 Bacillus subtilis WB800 其 γ-PGA 合成相關基因 pgsBCAE 是存在的,但此菌株卻非 γ-PGA 生產株。利用持續型及誘導型高效率的人工合成表現元件(SECS)導入宿主中並使其嵌於 pgsBCAE 基因的上游,使得 Bacillus subtilis WB800 轉形為 γ-PGA 生產株。轉形株中 Bacillus subtilis Dc8006 可穩定的生產高量 γ-PGA,且無須額外補充麩胺酸於培養基 medium A。
為了評估不同培養條件對生產 γ-PGA 的效果,進行 Plackett-Burman
因子設計實驗。檢查十二變量對 Bacillus subtilis Dc8006 其 γ-PGA 生產影響程度。基於統計分析得到預優化培養基,在優化培養基 medium PBD中發酵 γ-PGA 產量可達 35.2 克/升,高於原 medium A 培養基之 γ-PGA產量 1.5 倍。
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