Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/56748
標題: 以蛋白標靶技術及直接演化法改造地衣芽孢桿菌為高品質幾丁聚醣生產株
Construction of Bacillus Licherniformis Mutant Capable in High Quality Chitosan Production by Protein Targeting and Direct Evolution Techniques
作者: 葉娟美
關鍵字: 技術發展
Bacillus licheniformis
食品科技
地衣芽孢桿菌
蛋白標靶系統
幾丁聚醣?
protein targeting systems
摘要: 食品生技之產值為國內生物技術市場之冠,且年成長率也最高,是目前最為看好的明星產業。食品生物技術中,安全級微生物系統是大量生產精緻有用成分之重要關鍵技術。地衣芽孢桿菌為安全級高分泌能力菌株。本研究室已建立其基因操作條件及載體-宿主系統,並建立以最佳化人工合成表現控制元件構築之高表現系統。本計畫中, 將配合各種分泌元件及直接演化法,以改造訊息胜肽,獲得地衣芽孢桿菌最適分泌元件。建立地衣芽孢桿菌之幾丁聚醣酶高分泌系統;及幾丁聚醣酶大量純化製備流程。幾丁聚醣酶基因以Error Prone PCR法直接演化,改進其pH適應性、熱穩定性、pH穩定性、基質特異性及產物分子量。再改造為食品級質體。建立食品級地衣芽孢桿菌高分泌表現幾丁聚醣酶系統。地衣芽孢桿菌菌體表層展示系統則以細胞壁結合蛋白質融合先前最佳分泌表現元件建立,以固定化酵素模式,探討分解幾丁聚醣之條件。由於地衣芽孢桿菌本具幾丁分解酶能力,將以全菌株催化方式,直接利用幾丁質生產幾丁聚醣。本研究可提供新穎性幾丁聚醣酶生產系統,包括安全級分泌系統及表層展示系統;前者可用於大量純化或發酵液直接使用;後者可提供酵素固定化模式,簡化生產流程及成本。本模式未來可應用於各種高價值重組生技產品之製造。
The food biotechnology is considered as a potent high value industry in Taiwan. In recent report, the product yield of food biotechnology is highest among biotechnology market. To produce high value product in food biotechnology market, a key technique is the microbial expression system with safety, high expression level and ease of production process. Bacillus licheniformis is recognized as GRAS (generally recognized as safe) with high secretion capacity. We have developed the genetic techniques, and established high level expression system by using Synthetic Expression Control Sequences (SECSs). In this project, optimal secretion element will be engineered to obtain high secretion signals by testing several secretion signal peptides and engineered by error prone PCR. The high level secreted chitosanase will be purified by examining the optimal purification process. To obtain a proper characteristic chitosanase, the chitosanase is subjected to directed revolution by modified epPCR. Food grade selection marker nsr or nisI will be introduced into this expression system to achieve a GRAS grade chitosanase expression system of Bacillus licheniformis which provides a direct use of fermented product and simplified purification process without contaminant of antibiotics. A cell surface display system of Bacillus licheniformis is then developed by introducing the cell wall binding domain into the C-terminal of secretable chitosanase. The cell wall chitosanase-anchoring Bacillus licheniformis mutant is used to test as a chitosanase immobilization model and whole cell catalysis. This research provides potent tools for high productivity system of Bacillus licheniformis in food biotechnology.
URI: http://hdl.handle.net/11455/56748
其他識別: NSC97-2313-B005-015-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1650448&plan_no=NSC97-2313-B005-015-MY3&plan_year=97&projkey=PD9709-0081&target=plan&highStr=*&check=0&pnchDesc=%E4%BB%A5%E8%9B%8B%E7%99%BD%E6%A8%99%E9%9D%B6%E6%8A%80%E8%A1%93%E5%8F%8A%E7%9B%B4%E6%8E%A5%E6%BC%94%E5%8C%96%E6%B3%95%E6%94%B9%E9%80%A0%E5%9C%B0%E8%A1%A3%E8%8A%BD%E5%AD%A2%E6%A1%BF%E8%8F%8C%E7%82%BA%E9%AB%98%E5%93%81%E8%B3%AA%E5%B9%BE%E4%B8%81%E8%81%9A%E9%86%A3%E7%94%9F%E7%94%A2%E6%A0%AA
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