利用Streptomyces clavuligerus以進料批式操作下生產clavulanic acid之研究

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研究生：	龔建同
論文名稱：	利用Streptomyces clavuligerus以進料批式操作下生產clavulanic acid之研究
指導教授：	陳國誠黃世傑
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2004
畢業學年度：	92
語文別：	中文
論文頁數：	72
中文關鍵詞：	甘油進料
外文關鍵詞：	Streptomyces clavuligerus, clavulanic acid
相關次數：	點閱：50 下載：0
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Clavulanic acid是一種由Streptomyces clavuligerus發酵生產的β-lactamase抑制劑，醫療上經常合併使用clavulanic acid與amoxicillin。本論文利用批式與進料批式的操作，以S. clavuligerus進行clavulanic acid的發酵生產，探討大豆萃取液與clavulanic acid的前驅物甘油、ornithine、arginine對其生化合成的影響，並尋求最適的前驅物及其進料量，以及建立進料批式操作的方法。在搖瓶培養過程中，含有大豆萃取液(TKN=0.59 g/L)會有較佳的clavulanic acid產量，而進料批式操作的過程中，利用間歇進料的方式，將甘油持續進料到培養液內，其所生成clavulanic acid的最高濃度是未進行glycerol進料的2.3倍。同時進行甘油和ornithine或arginine的進料，所生成的clavulanic acid濃度分別是未進行glycerol進料的2.7、2.8倍。甘油的進料添加能夠提供clavulanic acid分子左側結構上β-lactam環的前驅物，而ornithine或arginine的添加則能提供clavulanic acid分子右側結構上C5-moiety的前驅物，這顯示甘油和ornithine或arginine能有效率地構成clavulanic acid。

Clavulanic acid is one of the fermentation products through secondary metabolism by Streptomyces clavuligerus. It is also a specific and irreversible inhibitor of a wide range of bacterial β-lactamases. Clavulanic acid has been used in human therapy for several years in combination with amoxicillin. The purpose of this study is to investigate the effects of different precursors and soy meal extract on the biosynthesis of clavulanic acid using S. clavuligerus in batch and fed-batch fermentations. A regularly intermittent feeding strategy was employed to obtain the maximum productivity of clavulanic acid when different feed medium was used to improve the production. The medium containing soy meal extract with the TKN concentration of 0.59 g/L was optimum. Intermittently feeding the medium containing glycerol to the shake flask cultures resulted in the maximum production of clavulanic acid being 2.3-fold higher than that with no additional feeding. Intermittent feeding simultaneously with glycerol and ornithine (or arginine) resulted in the maximum production of clavulanic acid being 2.7-fold (or 2.8-fold) higher than that with no feeding. The fed batch result indicates that glycerol is an important precursor of the β-lactam moiety(C-5,C-6, and C-7), whereas ornithine (or arginine) is an important precursor of C5-moiety. Both glycerol and ornithine (or arginine) exert a stimulation of the biosynthesis of clavulanic acid, and they are efficiently incorporated into clavulanic acid.

一、研究緣起    1
二、研究背景與目的    3
    2-1  Clavulanic acid之簡介    3
        2-1-1 β-Lactam抗生素    3
        2-1-2 Clavulanic acid的生化特性及其用途    4
    2-2  Clavulanic acid的生化合成與生產菌株    5
        2-2-1 Clavulanic acid 生產菌    5
        2-2-2 Clavulanic acid的生化合成路徑    6
        2-2-3 碳源對Clavulanic acid生化合成的影響    8
        2-2-2 氮源對Clavulanic acid生化合成的影響    12
    2-3  Clavulanic acid的發酵工程    18
        2-3-1 進料批式(Fed-batch)培養    18
    2-4  本實驗室過去的研究成果    20
    2-5  研究目的    21
三、實驗材料與方法    22
    3-1 實驗藥品    22
    3-2  Clavulanic acid生產菌    22
        3-2-1菌種    22
        3-2-2菌種活化與菌種保存    22
        3-2-3接種及培養方法    23
        3-2-4菌體量測法    24
    3-3 發酵槽培養及操作    24
        3-3-1  發酵槽設備    24
        3-3-2  發酵槽批式培養    25
        3-3-3  發酵槽進料批式培養    25
    3-4 分析方法    25
        3-4-1  Clavulanic acid濃度分析    25
        3-4-2  Glycerol濃度分析    27
        3-4-3  Ammonium濃度分析    27
        3-4-4總凱氏氮濃度分析    28
        3-4-5 胺基酸濃度分析    28
    3-5 實驗設備    28
四、結果與討論    30
    4-1大豆萃取液對clavulanic acid生化合成的影響    30
    4-1-1初始大豆粉萃取液濃度的影響    30
    4-1-2大豆粉萃取液的營養成份    30
    4-2胺基酸與ammonium ion對clavulanic acid生化合成的影響31
    4-2-1初始額外添加ornithine、arginine、ammonium ion
               的影響    31
    4-2-2初始額外添加不同濃度之ornithine、arginine的影
                響    32
    4-3 甘油與ornithine, arginine, ammonium ion, soy meal
         extrect合併進料的影響    33
    4-3-1甘油進料對clavulanic acid生化合成的影響    33
    4-3-2甘油與不同濃度ornithine或arginine合併進料的影
               響    36
    4-4溶氧的影響    37
    4-5發酵槽實驗    38
五、結論    41
六、參考文獻    43
七、圖表    53

                                

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