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Student Number 91324039
Author huei-chi Wu(吳惠琦)
Author's Email Address No Public.
Statistics This thesis had been viewed 1783 times. Download 1558 times.
Department Chemical and Materials Engineering
Year 2003
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title 探討溫度及剪切力對Actinoplanes sp.生產acarbose 之發酵影響
Date of Defense 2004-07-12
Page Count 55
Keyword
  • acarbose
  • Actinoplanes sp
  • temperature
  • Abstract 放線菌廣泛用於現今製藥抗生素,我們知道藉由Actinoplanes sp.生產acarbose,目前此藥物被一些有糖尿病第二型病人所使用,坊間也有用於在減重方面,不過成效還需更多的研究報告證實。
    一般認為培養溫度是影響菌體生長的主要因素。因此,大部分之研究是在最適合生長溫度下進行培養。但是文獻資料並沒有針對不同培養溫度及剪切力對發酵液的滲透壓與acarbose生成做相關研究。而在放線菌發酵中,培養液acarbose量是相當重要,具有治療糖尿病患者,我們利用發酵技術,如溫度或剪切力效應來增加菌體生長和acarbose生成,值得一提的是滲透壓也是作為探討之指標。
    本研究以攪拌式反應器來進行深層培養放線菌,利用生物技術發酵生產醣類醫藥原料acarbose。Acarbose主要為Glucobay(商品名)主成份,用於治療第二型糖尿病患者。Actinoplanes sp. 是目前研究發酵生產acarbose的主要菌種之一,在實驗中,我們探討了使用攪拌式發酵槽,在不同溫度(24℃、26℃、28℃)時,對放線菌生長和acarbose生成所造成的影響,結果有如下發現:
    1.在不同maltose濃度下,於搖瓶液態發酵中,可證實有最適當滲透壓範圍(150~250 mOsm/kg),另外於發酵槽液態發酵中,可知道適當滲透壓範圍(400~450mOsm/kg),同時對於不同溫度(24℃和28℃)隨時間改變的平均滲透壓範圍(350~450 mOsm/kg)。
    2.在不同培養溫度下,於發酵槽發酵中,發現在適當溫度範圍中(24℃和28℃)對acarbose生成和菌體生成等有一最適分配,在28℃時,菌體轉化率Yx/s = 0.1545、Yp/s=0.1342、Yp/x=0.79,在此溫度下是acarbose生成較多,但菌體量在80hr以後逐漸下降。當溫度為24℃時(Yp/x=0.3532),放線菌菌體維持穩定。
    3.不同發酵槽對放線菌液態發酵的影響,在放線菌液態發酵以4L攪拌式發酵槽比1.5L攪拌式有利。而在連續通氣和不連續通氣(如搖瓶)條件下,因為搖瓶實驗(150rpm、28℃)中由於沒有足夠的氧氣,以相同的發酵時間,在發酵過程中,使每一克菌絲體所能生產acarbose產量(Yp/x=0.4453)比在連續通氣中攪拌式(Yp/x=0.79)發酵槽低。
    4.了解acarbose生成穩定之問題。
    5.於實驗操作在溫度28 ℃下,有最佳之轉化率。
    Table of Content 目 錄
    中文摘要……………………………………………………………Ⅰ
    目錄………………………………………………………………….Ⅲ
    表索引……………………………………………………………….Ⅶ
    圖索引……………………………………………………………….Ⅷ
    第一章緒論
    1-1背景………………………………………………………….1
    1-2 研究目的……………………………………………………4
    第二章文獻回顧
    2-1放線菌之介紹(Actinoplanes sp.)…………………………5
    2-1-1放線菌的發現……………………………………………...5
    2-1-2放線菌的型態特徵與分類地位…………………………...6
    2-1-3 acarbose的發現與工業利用………………………………8
    2-1-4 acarbose的藥理研究………………………………………9
    2-1-5acarbose構造……………………………………………….10
    2-1-6放線菌生長環境分佈……………………………………...10
    2-1-7國內外之相關研究………………………………………...12
    2-2 放線菌(Actinoplanes)在生物技術產業之應用…………...15
    2-3在醱酵槽中,一些物理及化學因子對細菌生長型態及
    acarbose生成的影響………………………………………………15
    2-4生產方式與影響因子…………………………………………19
    2-5 acarbose生產動力學探討…………………………………….21
    第三章材料和方法
    3-1實驗材料………………………………………………………23
    3-1-1實驗菌株…………………………………………………….23
    3-1-2實驗藥品…………………………………………………….23
    3-1-3實驗儀器及其他設備……………………………………….24
    3-1-4實驗裝置…………………………………………………….25
    3-2實驗設計與方法………………………………………………26
    3-3實驗方法………………………………………………………26
    3-3-1菌種保存…………………………………………………….26
    3-3-2菌種活化…………………………………………………….26
    3-3-3培養基組成………………………………………………….27
    3-3-4操作條件…………………………………………………….27
    3-3-5分析方法…………………………………………………… 29
    1.醱酵液處理流程……………………………………………….29
    2.菌體量的測定………………………………………………….30
    3.葡萄糖殘量分析……………………………………………….30
    4.麥芽糖殘量分析……………………………………………….31
    5.滲透壓分析…………………………………………………….32
    6.醱酵液濃縮處理分析………………………………………….33
    7.acarbose分析…………………………………………………..33
    8.標準檢量線…………………………………………………….34
    3-4醱酵操作之實驗方法…………………………………………35
    3-4-1 針對不同濃度maltose對搖瓶醱酵實驗…………………. 35
    3-4-2不同轉速對acarbose生成和滲透壓之影響……………….36
    3-4-3培養溫度對acarbose和滲透壓之影響……………………..37
    第四章結果與討論
    4-1放線菌菌種觀察………………………………………………38
    4-1-1菌體在固態培養基上外觀………………………………….38
    4-1-2搖瓶培養…………………………………………………….39
    4-1-3反應器外觀與實驗架構…………………………………….40
    4-2搖瓶實驗………………………………………………………41
    4-3醱酵槽實驗……………………………………………………42
    4-4不同轉速之發酵槽實驗………………………………………43
    4-5溫度對acarbose液態醱酵之影響……………………………45
    4-6 acarbose的生成對滲透壓分析的結果……………………….48
    第五章結論與建議……………………………………………………51
    參考文獻………………………………………………………………..53
    表索引
    表2-1、Actinoplanes之形態特徵………………………………………...7
    表2-2、Actinoplanes之形態與物理特徵………………………………7
    表2-3、Actinoplanes之微觀生理特徵…………………………………8
    表2-4、比較acarbose分析方法………………………………………...12
    表2-5、工業發酵上常用的碳源和氮源……………………………….20
    表2-6、八種主要培養基和生化功能以及生長所需的濃度…………20
    表3-1、實驗藥品………………………………………………………23
    表3-2、Actinoplanes sp.固態培養基組成……………………………..28
    表3-3、Actinoplanes sp.種瓶培養基組成……………………………..28
    表3-4、Actinoplanes sp.主醱酵槽之培養基組成……………………...28
    表4-1、以搖瓶醱酵探討不同麥芽糖對acarbose影響之結果………..41
    表4-2、添加不同濃度maltose之醱酵情形…………………………….43
    表4-3、比較搖瓶與發酵槽之菌種和acarbose生成變化…………….44
    表4-4、攪拌式醱酵槽探討溫度對acarbose生成影響之結果………...47
    表4-5、隨著不同濃度的麥芽糖其醱酵出之滲透壓值………………48表4-6、隨著醱酵時間下之平均滲透壓值…………………………….50
    圖索引
    圖2-1、Pseudo-oligosaccharidicα-glucosidase inhibitors of the
    general formula…………………………………………………………...9
    圖2-2、acarbose化學結構………………………………………………10
    圖2-3、acarbose 作用機制……………………………………………..11
    圖2-4、Postulated pathway of acarbose biosynthesis in Actinoplanes
    sp. SE50…………………………………………………………………14
    圖2-5、amino sugar 結構……………………………………………...18
    圖2-6、工業上放大製造acarbose……………………………………...21
    圖3-1、發酵槽裝置圖………………………………………………….25
    圖3-2、發酵液處理流程………………………………………………..29
    圖3-3、葡萄糖圖譜…………………………………………………….30
    圖3-4、麥芽糖圖譜…………………………………………………….31
    圖3-5、微量滲透壓分析儀……………………………………………32
    圖3-6、acarbose圖譜………………………………………………….34
    圖3-7、acarbose之標準檢量線……………………………………….35
    圖4-1、固態培養基生長情形…………………………………………..38
    圖4-2、在攪拌式醱酵槽中生長前期之情形…………………………40
    圖4-3、在攪拌式醱酵槽中生長後期之情形…………………………..40
    圖4-4、搖瓶實驗中不同濃度maltose對每一克菌體所產生之acarbose轉化率Yp/s的影響……………………………………………………..41
    圖4-5、添加20g/l的maltose發酵動力曲線圖……………………….42
    圖4-6、添加40g/l的maltose發酵動力曲線圖………………………43
    圖4-7、不同轉速對每一克菌體所產生之acarbose之影響………….44
    圖4-8、轉速對放線菌體轉化率Yx/s的影響…………………………45
    圖4-9、轉速對每一克菌體所產生之acarbose之影響………………45
    圖4-10、28℃醱酵動力曲圖…………………………………………..46
    圖4-11、24℃醱酵動力曲線圖…………………………………………47
    圖4-12、溫度對每一克菌體所產生之培養液acarbose之影響……..48
    圖4-13、於搖瓶下,隨著maltose濃度的不同滲透壓之變化情形…49
    圖4-14、於發酵槽下,隨著maltose濃度其醱酵出之平均滲透壓
    值之變化情形…………………………………………………………..49
    圖4-15、隨著不同溫度之發酵時間下之平均滲透壓值..........50
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  • Chin-Hang Shu(徐敬衡)
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    Date of Submission 2004-07-16

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