臺灣博碩士論文加值系統

木質醋酸菌(Acetobacter xylinum)所產生的細菌纖維素(Bacterial cellulose, BC)，是一種由生物聚合的高分子具有生物可分解性、親水能力、超細微纖維等優點，已被廣泛應用於食品工業，作為食品添加物，如保水劑、食品增稠劑、安定劑，或作為高纖食品之原材料，為極具潛力的一項商品。本研究藉由遺傳工程技術將Lipase基因或綠色螢光蛋白基因轉殖片段轉殖(Cloning)於木質醋酸菌中。轉殖的木質醋酸菌能表現這些外源蛋白於細胞表面，將有利於未來食品或其他工業之用途。

Bacterial cellulose (BC) produced from Acetobacter xylinum, is polysaccharide with unique properties including biodegradation, high water holding capacity, and a fine fiber network. With the characteristics, BC has been widely applied to food engineering as food additives, thickener, stabilizer, and high fiber materials. It is promosing commercial materials. In this study, a transformed Acetobacter xylinum carried a lipase gene or green fluorescent protein gene was generated. These foreign proteins were successfully expressed by transformed Acetobacter xylinum and were located in the surface of cells, suggesting a promising application to food or other industries.

誌謝--------------------------------------------------------------------------- i
中文摘要--------------------------------------------------------------------------- ii
英文摘要--------------------------------------------------------------------------- iii
目錄--------------------------------------------------------------------------- iv
表目錄--------------------------------------------------------------------------- vi
圖目錄--------------------------------------------------------------------------- vii
第一章緒論--------------------------------------------------------------------- 1
第二章文獻回顧--------------------------------------------------------------- 2
第一節木質醋酸菌之簡介--------------------------------------------------- 2
一木質醋酸菌之特性--------------------------------------------------- 2
二木質醋酸菌生產細菌纖維素合成之機制------------------------ 4
三木質醋酸菌轉形之相關研究--------------------------------------- 8
四影響細菌纖維素生長之因素--------------------------------------- 9
五細菌纖維素之應用--------------------------------------------------- 11
第二節外源蛋白簡介--------------------------------------------------------- 15
一螢光蛋白--------------------------------------------------------------- 15
二脂解酵素(Lipase)----------------------------------------------------- 16
第三章研究方法--------------------------------------------------------------- 19
第一節實驗流程圖------------------------------------------------------------ 19
第二節實驗材料--------------------------------------------------------------- 20
一菌株--------------------------------------------------------------------- 20
二質體--------------------------------------------------------------------- 20
三引子primers----------------------------------------------------------- 21
四藥品--------------------------------------------------------------------- 22
五DNA 純化套件組---------------------------------------------------- 23
第三節儀器設備--------------------------------------------------------------- 24
第四節實驗方法--------------------------------------------------------------- 25
一菌株的保存與培養--------------------------------------------------- 25
二萃取質體DNA-------------------------------------------------------- 25
三萃取Genomic DNA-------------------------------------------------- 26
四膠體純化PCR產物--------------------------------------------------- 27
五質體與PCR 產物之Digestion-------------------------------------- 27
六質體與PCR 產物之Ligation -------------------------------------- 28
七轉形作用--------------------------------------------------------------- 28
八重組菌株的篩選與培養--------------------------------------------- 30
九西方墨點法分析------------------------------------------------------ 31
十酵素活性分析--------------------------------------------------------- 31
十一纖維分解酵素之作用------------------------------------------------ 32
第四章結果與討論------------------------------------------------------------ 33
第一節轉殖A. xylinum 表現綠色螢光蛋白------------------------------- 33
一建構pET22b 含綠色螢光蛋白基因質體與A. xylinum 的轉殖33
二以不同IPTG 濃度進行外源蛋白誘導---------------------------- 36
三培養方式對轉殖A.xylinum 生產綠色螢光蛋白的影響-------- 38
第二節轉殖A. xylinum 與脂解酵素Lipase 之表現---------------------- 40
一建構pET22b 含與脂解酵素Lipase 基因質體與
A. xylinum 的轉殖----------------------------------------------------- 40
二轉殖菌株質體快速篩選--------------------------------------------- 45
三重組菌株之蛋白質表現--------------------------------------------- 47
四轉殖A. xylinum Lipase 的活性分析------------------------------- 49
第五章結果與討論------------------------------------------------------------ 57
參考文獻--------------------------------------------------------------------------- 58
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