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研究生:曹子辰
研究生(外文):Tzu-Chen Tsao
論文名稱:用環形桿菌TTU-24在五公升醱酵槽中生產環狀糊精合成酵素的最適化
論文名稱(外文):Optimization of Production of Cyclodextrin Glucanotransferase by Bacillus circluans TTU-24 in a 5-liter Jar Fermentor
指導教授:許垤棋
指導教授(外文):Dey-Chyi Sheu
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:英文
論文頁數:160
中文關鍵詞:環狀糊精合成酵素環形桿菌溶氧傳輸率醱酵實驗設計
外文關鍵詞:cyclodextrin glucanotransferaseBacillus circluansoxygen transfer ratefermentationfactorial design
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本研究是用環形桿菌(Bacillus circluans)TTU-24,在五公升醱酵槽中生產環狀糊精合成酵素(CGTase),探討其最適的攪拌速率與通氣量以達到酵素的最大產量。洋菜平板培養基以含有1%玉米澱粉、2%酵母抽出物、1.5%洋菜和1%碳酸鈉的配方,最適合環形桿菌TTU-24孢子的萌發和生長以及後續的環狀糊精合成酵素的醱酵生產。比較各種澱粉當作液態培養基之碳源,其中以玉米澱粉與可溶性澱粉(soluble starch)為較佳的碳源。樹薯澱粉(cassava starch)經a-amylase水解後,再用擠壓機加工當作培養基之碳源,對環狀糊精合成酵素的生產有正面的作用。醱酵槽種菌生長至對數期,其細胞密度介於(OD600nm) 0.4-0.6之間為最適當的接種時機。氧氣傳輸對於環形桿菌 TTU-24的酵素生產有很大的影響,攪拌速率與通氣量的交互作用的影響頗為顯著。經由實驗設計(factorial design)規劃醱酵槽的攪拌速率與通氣量,再用GLM的統計分析,得到最適酵素生成的通氣和攪拌的趨勢,並且用central composite design (CCD)求出最適化攪拌速率與通氣量。經過一系列的實驗,得到最適的攪拌速率為650 rpm,最適的通氣量為 2.1 vvm,所得到的環狀糊精合成酵素(CGTase)的最大生成量約為14,000 unit/ml。溶氧傳輸係數(dissolved oxygen transfer coefficient, KLa)可當作攪拌速率與通氣量的交互作用的參照,經由動態的溶氧濃度測定法得到最適化的攪拌速率與通氣量下的KLa值為212.5 h-1。

An optimum production of cyclodextrin glucanotransferase (CGTase) by Bacillus circulans TTU-24 in alkaline medium using a 5-liter jar fermentor under the conditions of various agitation aerates and aeration rates was investigated. A plate agar medium consisting of 1% corn starch, 2% yeast extract, 1.5% agar and 1% Na2CO3 was the optimum medium for the endospore germination and the cell growth of this microorganism. Corn starch and soluble starch were better carbon sources for the production of CGTase in comparison with other starches and a press extrusion treatment after partial hydrolysis by a-amylase had positive effect on cassava starch being used as carbon source for the same purpose. An inoculum thriving in log phase with OD600 of 0.4-0.6 was most suitable for the culture in jar fermentator. Oxygen transfer rate, an interaction of agitation and aeration in jar fermentor, influenced greatly on the production of CGTase by Bacillus circulans TTU-24. Optimum rates of agitation and aeration were obtained based on factorial designs and central composite designs (CCD). Through a series of experiments, a maximum CGTase activity of 14,000 unit/ml was produced under an optimum condition with an agitation rate of 650 rpm and an aeration rate of 2.1 vvm. According to a dynamic determination of dissolved oxygen, the optimum KLa value for the production of CGTase by Bacillus circulans TTU-24 was to be 212.5 hr-1.

CONTENTS
PAGE
ABSTRACT ……………………………………………………………….. I
CHINESE ABSTRACT ………………………………………………. II
CONTENTS ……………………………………………………………….. IV
LIST OF TABLES ………………………………………………………. VII
LIST OF FIGURES …………………………………………………….. IX
CHAPTER 1 INTRODUCTION ………………………………. 1
CHAPTER 2 PAPER REVIEW ………………………………... 3
2.1 The Properties of Cyclodextrins ( CDs ) ………..………………… 3
2.2 The Application of Cyclodextrins ( CDs ) ………………………. 6
2.3 Intra- and Intermolecular Transglycosylating Reaction of the
Bacterial Cyclodextrin Glucanotransferase ( CGTase ) …….. 8
2.4 The Production of Bacterial CGTase ………..…………………… 12
2.5 The Structure of the Bacterial CGTase …………..……………… 17
2.6 The Effects of the Dissolved Oxygen Concentration ………….. 21
CHAPTER 3 MATERIALS AND METHODS ……….…. 23
3.1 Materials ………………………………………………………………… 23
3.1.1 Culture Media and Chemicals …..……………………… 23
3.1.2 Microorganism ……………………………………………... 23
3.2 The Optimum Plate Agar Medium for the Bacterium ……… 25
3.3 The Effects of Various Starches on the Production of CGTase 25
3.4 The Effects of Various Extruded Starches on the Production
of CGTase ……………………………………………………………….. 27
3.5 The Optimum Timing of Inoculum ……….……………………….. 28
3.6 The Optimum Rates of Agitation and Aeration …...…………… 29
3.7 Analytical Methods …………...……………………………………….. 30
3.7.1 Cell Density …………...……………………………………….... 30
3.7.2 pH Value …………...…………………………………………….. 30
3.7.3 Dissolved Oxygen Value ……..……………………………….. 30
3.7.4 The CGTase Activity ……...…………………………………... 31
3.7.5 The Dissolved Oxygen Transfer Coefficient ( KLa ) ....... 34
3.8 The Factorial Design …….……………………………………………. 34
3.9 The Central Composite Design ( CCD ) ………………………….. 35
3.10 Computer Hardware and Software ……..……………………….. 36
Chapter 4 RESULTS AND DISCUSSION ………………… 38
4.1 The Optimum Plate Agar Medium for the Bacterium ……….. 38
4.2 The Effects of Various Starches on the Production of CGTase 41
4.3 The Effects of Various Extruded Starches on the Production
of CGTase ……………………………………………………………….. 53
4.4 The Optimum Timing of Inoculation of Seed Culture …………. 62
4.5 The Optimum Agitation Rate and Aeration Rate …..………….. 66
4.5.1 The First Step of Screening Design ……….……………….. 66
4.5.2 The Second Step of Screening Design ……...……………. 75
4.5.3 The First Step of Searching the Design of Optimal Point 83
4.5.4 The Second Step of Searching the Design of Optimal
Point …………………….…………………………………….…… 91
CHAPTER 6 CONCLUSION …………………………………… 102
REFERENCES …………………………………………………………… 104
APPENDICES …………………………………………………………... 114
Appendix 1: Advantech Genie Software ………...………………….… 114
Appendix 2: SAS Program …………………………………..…………… 123
Appendix 3: Time Course of Batch Fermentation ………….…….… 128

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