臺灣博碩士論文加值系統

Clavulanic acid為β-lactamase抑制劑，常與青黴素類(penicillins)抗生素合併使用來克服病原菌的抗藥性。clavulanic acid與amoxicillin的合併藥方是目前在醫療上被廣泛的使用。
本論文利用S. clavuligerus進行clavulanic acid的發酵生產，在批式和進料批式的操作，探討前培養的特性對clavulanic acid生產的影響，並在進料批式培養中，探討glycerol在培養液中最適維持濃度及檢討glycerol與ammonium ion的進料對clavulanic acid生化合成的效應。
前培養的菌體特性與生長形態會影響後續clavulanic acid的發酵生產。以不同的接種菌齡與接種比例進行clavulanic acid的批式與進料批式的發酵培養，得到最適的接種菌齡及接種比例分別為28小時與10％。以間歇進料的方式進料glycerol進行搖瓶進料批式培養，探討不同glycerol在培養液中最低維持濃度及其進料量，得知glycerol在培養液中的濃度變化維持在1∼5 g/l，其clavulanic acid產量最佳。不論是否有調控培養液中的pH或者初始添加ornithine與否，以glycerol與ammonium ion合併進料的結果皆顯示，微量的添加ammonium ion (5 mg)的clavulanic acid產量比單獨glycerol進料的培養的增加約15∼30％，之後隨著ammonium ion進料量的增加，產量會受到抑制。以初始含ornithine 5 mM，每次進料0.25 g glycerol和ammonium ion 5 mg，並控制pH的進料批式培養所得到的clavulanic acid產量最佳，為批式培養的2.74倍。

Clavulanic acid is 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 effect of inoculum quality on the production of clavulanic acid was first investigated in batch or fed-batch flask cultures with S. clavuligerus. A methodology to obtain an optimal concentration maintaining range for glycerol in the culture was developed to enhance the clavulanic acid production. Furthermore, the effect of intermittently feeding both glycerol and ammonium ion on the biosynthesis of clavulanic acid was also investigated.
The inoculum quality and the morphology did influence the clavulanic acid production. The optimal age and size of inoculum for the production of clavulanic acid in batch and fed-batch flask culture was 28 hrs and 10%, respectively. The optimal concentration range of glycerol could be maintained low in the culture, and the concentration range for clavulanic acid biosynthesis were 1~5 g/l. Whatever we try to control the pH of the cultures or initially add ornithine to the cultures, intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg in shake flask cultures resulted in the maximum production of clavulanic acid that was 15~30% higher than that with only feeding glycerol culture. Subsequently, the biosynthesis of clavulanic acid was inhibited due to the increasing amount of ammonium ion continuously added. The highest maximum production of clavulanic acid could be obtained under the conditions: initially concentration of ornithine of 5 mM, and simultaneously and intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg with the pH controlled in the culture. It was 2.74-fold higher than batch culture.

目 錄
一、研究緣起 1
二、研究背景與目的 2
2-1β-lactam抗生素簡介 2
2-1-1β-lactam抗生素之分類 2
2-1-2β-lactamase的抗藥性機制 2
2-2 Clavulanic acid簡介 4
2-2-1 Clavulanic acid生產菌 4
2-2-2 Clavulanic acid生化特性及其用途 5
2-3 Clavulanic acid生化合成路徑 6
2-4 前培養活性對發酵生產的影響 9
2-5 碳源、氮源進料對發酵生產的影響 11
2-5-1基質進料對發酵生產的影響 11
2-5-2 Ammonium進料的影響 12
2-6 Fed-batch培養簡介 13
2-6-1 Fed-batch培養原理及其優缺點 13
2-6-2 Fed-batch培養在抗生素生產的應用 13
2-7 先前研究結果 14
2-8 研究目的 15
三、實驗材料與方法 16
3-1 實驗藥品 16
3-2 Clavulanic acid生產菌 16
3-2-1 菌種 16
3-2-1 菌種活化與菌種保存 16
3-3 接種及培養方法 17
3-4 分析方法 18
3-4-1菌體量測法 18
3-4-2 Clavulanic acid濃度分析 18
3-4-3 Glycerol濃度分析 20
3-4-4 Ammonium濃度分析 20
3-5 實驗設備 21
四、結果與討論 22
4-1 接種菌體活性對clavulanic acid生化合成的影響 22
4-1-1 前培養的生長曲線 22
4-1-2 不同前培養時間對批式生產培養之影響 22
4-1-3 不同接種比例對批式生產培養之影響 25
4-1-4 不同前培養時間對進料批式生產培養之影響 26
4-2 Glycerol進料對clavulanic acid生化合成的影響 27
4-2-1 Glycerol最低維持濃度及其進料量的影響 27
4-3 Glycerol與ammonium ion同時進料對clavulanic acid 生化合成的影響 30
4-3-1 Ammonium ion合併進料對clavulanic acid生化 合成的影響 30
4-3-2 控制培養液的pH之影響 33
4-3-3 初始添加ornithine之影響 34
五、結論 36
六、參考文獻 38
七、圖表 46

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