利用基因重組巨型桿菌生產纖維分解酵素EglA之研究

Production of cellulase EglA by recombinant Bacillus megaterium

吳佳恩(Chia-En Wu)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10389

摘要

植物細胞壁之主要成分纖維素，為自然界蘊藏最豐的碳源與多醣類，亦為最充裕的再生資源。由於纖維素結構緊密，其內含的醣類難以利用，須以化學或生化反應的方式將其水解，以提高纖維素於生物能源再利用率，而纖維分解酵素扮演了極重要的角色。
　　纖維分解酵素為一群水解酵素的總稱，可經協同(synergic)作用將纖維素分解，目前已知具有纖維素分解能力的生物包括細菌、真菌、原生動物等。其用途相當廣泛，已應用於眾多產業上，主要為食品與釀酒、動物飼料、紡織與衣物清洗、造紙工業及農業，其他用途包括：廢棄物處理、製藥工業、基因工程及污染防治等。目前已有多種同源(homologous)或異源(heterologous)表達系統，如細菌、酵母菌及絲狀真菌等，可生產出具功能性之纖維分解酵素。
　　本研究以巨型桿菌(Bacillus megaterium)表達Piromyces rhizinflatus之內切型纖維分解酵素(endoglucanase, EglA)。以T7啟動子替代商品化選殖載體pWH1520之木糖啟動子，並導入巨型桿菌β-澱粉水解酵素之訊息胜肽，以期得到強力啟動子且具外泌能力之載體，更進一步比較不同表達質體表現纖維分解酵素EglA之效果。
　　目前已完成外泌性載體pWH1520-S、T7 RNA聚合殖載體pWH1520-T7R之建構，表現質體pWH1520-C，亦完成T7啟動子之釣取，待接入目標基因eglA後，即可進行纖維分解酵素之生產及比較不同表達系統之功效。可成功地於巨型桿菌B. megaterium WH1520-C中穩定表達纖維分解酵素EglA，其酵素活性達5 U/mL；亦可於B. megaterium WH1520-T7R中表達T7 RNA聚合酶。

關鍵字

巨型桿菌 ； T7 RNA聚合酶蛋白質外泌 ； 異源表達 ； 纖維分解酵素

並列摘要

Cellulose, the major component of plant cell walls, is the most abundant renewable biomass in nature and a sustainable clean resource for energy. Cellulase is responsible for the first step of degradation of cellulose. The application potential of cellulase includes food, brewery and wine, animal feed, textile and laundry, pulp and paper, as well as agricultures. Several homologous or heterologous expression systems, such as bacteria, yeasts or filamentous fungi, have been developed for production of cellulase.
Bacillus megaterium has several advantages over E. coli for a broad industrial use in the production of recombinant proteins. First, ease of culture, non-pathogenic and no inclusion body. Second, there is no alkaline proteases. Last, the recombinant plasmids in B. megaterium is stable structurally and segregationally.
In this study, we described the establishment of a B. megaterium based recombinant protein production and export system for the cellulase EglA from Piromyces rhizinflatus. We modified the B. megaterium by adapting E. coli T7 system for over-production of proteins and inserting a signal peptide from homologous β-amylase gene for protein secretion. The goal of this study is to develop an alternative expression system which could secret the desired protein into the culture supernatant. We have constructed secretory cloning vector pWH1520-S, xylose-inducible T7 RNA polymerase vector pWH1520-T7R, expression plasmid pWH1520-C. We also cloned T7 promoter gene and inserted eglA gene downstream. We have successfully express cellulase EglA in B. megateirum WH1520-C, and 5 Units/mL of crude extraction were measured.

並列關鍵字

Bacillus megaterium ； T7 RNA polymerase ； secretion ； heterologous expression ； cellulase

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