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研究生:王詩涵
研究生(外文):Shih-Han Wang
論文名稱:以碎米水解液為碳源生產海藻糖合成酶之研究
論文名稱(外文):Production of Trehalose Synthase Using Broken Rice Hydrolysate as Carbon Source
指導教授:方繼方繼引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:91
中文關鍵詞:海藻糖合成酶碎米水解液基因重組大腸桿菌
外文關鍵詞:trehalose synthasebroken rice hydrolysaterecombinant Escherichia coli
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碎米為碾米過程中的副產物,其價格低廉。本研究利用低經濟價值之碎米為原料,以酵素進行液化糖化作用,將所獲得的高葡萄糖含量水解液為碳源,調整適當濃度並加入其他營養源作為培養基,培養含有Picrophilus torridus trehalose synthase 基因片段的Escherichia coli RGB (DE3) (E. coli PTTS in RGB) 重組大腸桿菌,進行海藻糖合成酶的生產。
100-L發酵槽大量製備碎米水解液時,8公斤碎米粉末加入20 公升 R.O. 水和100毫升液化澱粉酵素(SPEZYME® AA),於 95℃、150 rpm下作用90分鐘後,葡萄糖、麥芽糖和麥芽三糖的含量分別為17.01、39.43和34.01 mg/ml ;待溫度降溫至60℃後,加入100毫升糖化澱粉酵素(OPTIMAX® 7525 HP),反應120分鐘,最終可得含218.14 mg/ml葡萄糖的碎米水解液。
以碎米水解液(BRH)為碳源、peptone為氮源,經反應曲面法探討最適的碳氮源比例為碎米水解液3.411%、peptone 0.705%。以此條件,並用150 mM pH 8磷酸鈉緩衝溶液配製培養基,於接菌量O.D.600 2.0、27℃、110 rpm下培養48小時後,離心破菌,可得海藻糖合成酶粗酵素液,酵素活性為45.07±1.43 U/ml,與以LB培養得到之結果相近。
由BRH和LB培養獲得之海藻糖合成酶經過純化後,其酵素特性皆為,最適作用溫度為45℃,並於60℃以下有很好的酵素穩定性;最適作用pH值為pH 6.0,於pH 5.0仍有高酵素活性(約80%),在pH 6、7、8都有很好的酵素穩定性。
Broken rice is a low price byproduct from rice milling process. In this study, broken rice was used as a potential carbon source for trehalose synthase production by recombinant Escherichia coli RGB (DE3). Hydrolysate was obtained from enzyme saccharification. Broken rice hydrolysate (BRH) with high glucose content was used as a carbon source in a medium containing other nutrient to produce trehalose synthase by cultivating recombinant E. coli RGB (DE3) barring Picrophilus torridus trehalose synthase gene fragment (E. coli PTTS in RGB).
In a 100-L fermentor, 8kg broken rice flour, 20 L R.O. H2O, and 100 ml amylase (SPEZYME® AA) was reacted at 95℃, 150 rpm for 90 min. After reaction, the contents of glucose, maltose and maltotriose was 17.01, 39.43 and 34.01 mg/ml, respectively. When temperature decreased to 60℃,100 ml glucoamylase (OPTIMAX® 7525 HP) was added for further hydrolysis. After reaction for 120 min, broken rice hydrolysate with 218.14 mg/ml glucose was obtained.
Response surface methodology (RSM) was applied to evaluate the optimal concentration of BRH and peptone to produce trehalose synthase by E. coli RGB (DE3). The results showed that 3.411% BRH and 0.705% peptone support the highest trehalose synthase activity production to a concentration of the medium. Under this medium composition and mixed with 150 mM pH 8 sodium phosphate buffer, 45.07 ± 1.43 U/ml of crude trehalose synthase activity was obtained after 48 hr of cultivation at the inoclum size of O.D.600 2.0 at 27℃and 110 rpm. The result was closed to trehalose synthase activity produced by the same microorganism that cultivated in LB broth.
The characteristics of the purified trehalose synthase produced by E. col RGB (DE3), which cultivated in LB or BRH were the same. The optimum reaction temperature was found to be 45℃ and with good stability at 60℃. The optimum reaction pH value was pH 6.0 and about 80% of enzyme activity was obtained at pH 5.0. This enzyme was found to have good pH stability at pH 6, 7, and 8.
摘要 I
Abstract II
第一章 前言 1
第二章 文獻回顧 2
一、稻米之介紹 2
二、酵素於澱粉工業上的應用 5
(一)α-amylase 5
(二)Pullulanase 7
(三)Glucoamylase 7
三、關於海藻糖及海藻糖合成酶 7
(一)海藻糖的發現 7
(二)海藻糖的結構及特性 8
(三)海藻糖的應用 8
(四)海藻糖的生產 8
(五)海藻糖合成酶(trehalose synthase) 14
四、關於基因重組大腸桿菌 17
(一)實驗菌株介紹 17
(二)基因重組大腸桿菌的培養 21
(三)以乳糖做為誘導劑表現外來蛋白質 24
五、反應曲面法 25
(一)反應曲面法之應用 25
(二)反應曲面法之原理 26
(三)二水準因子設計(Two-level factorial design) 27
(四)中心混層設計(Central composite design) 27
(五)正則分析(Canonical Analysis) 28
(六)脊形分析 28
第三章 實驗大綱 30
第四章 材料與方法 31
一、實驗材料 31
(一)原料 31
(二)實驗菌株 31
(三)培養基 31
(四)抗生素 31
(五)酵素 32
(六)純化海藻糖合成酶使用之藥品 32
(七)SDS-PAGE使用之藥品 32
(八)化學藥劑 34
(九)儀器設備 34
(十)套裝軟體 36
二、實驗方法 36
(一)以三角錐瓶探討液化澱粉酵素作用條件 36
(二)糖化澱粉酵素作用條件 37
(三)以100公升發酵槽製備碎米水解糖化液 37
(四)菌種保存及活化 38
(五)菌種生長曲線製作及酵素活性比較 38
(六)水解液(BRH)濃度對於菌體之影響 39
(七)10% 碎米水解液添加不同氮源種類對菌體之影響 39
(八)10% 碎米水解液添加不同peptone濃度之影響 39
(九)不同接菌量之影響 39
(十)不同培養階段添加乳糖做為誘導劑對菌體之影響 40
(十一)不同pH值緩衝溶液配製培養基對菌體之影響 40
(十二)不同緩衝溶液濃度對菌體之影響 40
(十三)以反應曲面法探討最適碳氮源比例 40
(十四)海藻糖合成酶特性分析 41
(十五)分析方法 41
第五章 結果與討論 47
ㄧ、碎米水解液的製備 47
(一)液化澱粉酵素作用條件探討 48
(二)糖化澱粉酵素作用條件 54
(三)以100-L發酵槽進行碎米水解液製備 56
二、以碎米水解液培養E. COLI PTTS IN RGB之探討 58
(一)水解液(BRH)濃度對於菌體之影響 60
(二)10% 碎米水解液添加不同氮源種類對菌體之影響 60
(三)10% 碎米水解液添加不同peptone濃度之影響 63
(四)不同接菌量之影響 65
(五)於不同培養時間添加乳糖的影響 67
(六)不同pH值緩衝溶液的影響 67
(七)不同緩衝溶液濃度對菌體之影響 72
三、以RSM探討碳氮源添加比例 74
四、海藻糖合成酶特性分析 78
(一)溫度對海藻糖合成酶活性及穩定性之影響 78
(二)pH值對海藻糖合成酶活性及穩定性之影響 78
五、使用原料價格估算 82
第六章 結論 83
第七章 未來展望 84
第八章 參考文獻 85
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