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|標題:||Investigation on the cultivation conditions of recombinant E. coli in penicillin G acylase production
|作者:||黃宏彰||關鍵字:||盤尼西林醯胺酵素;penicillin acylase;基因重組大腸桿菌;recombinant E. coli||出版社:||化學工程學系||摘要:||
盤尼西林醯胺酵素（penicillin G Acylase、簡稱PAC酵素），其功能為進行盤尼西林Penicillin G之水解，以生產抗生素原料藥之六青黴素酸 (6-aminopenicillanic acid, 6APA)。以大腸桿菌生產PAC酵素為工業上常用之方式，由於PAC酵素係E.coli之胞內酵素，為了達到PAC酵素的高產率，本研究構想於醱酵過程中，進行高密度細胞醱酵培養。
在探討溶氧因子對菌體生長與生產的實驗中發現，其菌體生長與酵素活性會因溶氧不足的環境下，菌體生長會受到抑制，其所生產之PAC酵素活性會隨時間而衰減。在低溶氧的條件下培養（5%），其酵素比活性即不因菌體的增加而下降，但其酵素比活性平均為162 IU/g-DCW、最高酵素體積活性僅達1.57 IU/ml。若以足夠的溶氧（20%及40%）狀態下進行培養，則有助於菌體生長與酵素生產，其菌體濃度可達11.29 g-DCW/L、最高酵素體積活性2.41 IU/ml、平均酵素比活性提升至206 IU/g-DCW。
當以不同銨離子濃度條件培養下，細胞菌體濃度與酵素活性會隨著培養基中銨離子濃度的增加而增加，當培養基中銨離子濃度為0.5 %時，其在醱酵末期會因氮源不足，導致醋酸累積至3%，並造成菌體生長之抑制；故其菌體濃度僅為10.12 g DCW/l，酵素體積活性為2.40 IU/ml。若將銨離子濃度增至1.5 %時，醋酸之累積為1.02 %，而其菌體濃度為17.58 g-DCW/l，酵素體積活性為3.42 IU/ml。當銨離子濃度為2.5 %時，其菌體濃度為16.09 g-DCW/l，酵素之體積活性為3.37 IU/ml。
為了進一步提高菌體及酵素活性產量，本實驗以批次饋料進行醱酵培養。然而，細胞的生長速率會因代謝性副產物醋酸的累積而受到限制，其生長速率由3.367 DCW/hr下降至0.965 DCW/hr。當以細胞回流進行培養，則有效地改善代謝性副產物醋酸的累積，其細胞生長速率提升至2.62 g-DCW/hr，細胞濃度達28.1 g-DCW/l，酵素的體積活性由4.76 IU/ml提升至5.92 IU/ml。
Penicillin G acylase (PAC) hydrolyzes penicillin G to 6-aminopenicillanic acid (6-APA), which is used in the semi-synthetic antibiotics. Recombinant E. coli is preferentially used for production of PAC enzyme in industry. Because E. coli produces PAC enzyme intracellularly, in order to enhance the PAC productivity, high cell density cultivation is required. In this study, high cell density cultivation of E.coli was investigated in fermentation process.
In studying the effect of dissolved oxygen on cell growth and production, it showed that cell growth was inhibited and PAC activity level decreased with time in insufficient dissolved oxygen condition. However, in low dissolved oxygen condition (5%), the specific enzyme activity would not decrease with the increase of the cell concentration, it was maintained at about 162 IU/g-DCW with a maximum volumetric enzyme activity of 1.57 IU/ml. In sufficient dissolved oxygen situation (20%~40%), the cell concentration and maximum volumetric enzyme activity were 11.29 g-DCW/L and 2.41 IU/ml respectively with a specific enzyme activity of 206 IU/g-DCW.
In studying of ammonia concentration effect, it showed that cell growth and enzyme activity increased with the increase of ammonium ion concentration (from range of 0.5%~1.5%). In situation that ammonium ion concentration was 0.5% in the medium, acetic acid accumulated to 3% and cell growth was inhibited due to insufficient nitrogen source in the fermentation medium. In this case, the cell concentration and enzyme activity reached 10.12 g DCW/l and 2.40 IU/ml respectively. When the ammonium ion concentration increased to 1.5%, acetic acid concentration decreased to 1.02 %, cell concentration and enzyme activity rised to 17.58 g DCW/l and 3.42 IU/ml respectively. As the ammonium ion concentration is 2.5%, the cell concentration and volumetric enzyme activity showed 16.09 g DCW/l and 3.37 IU/ml respectively.
For further increasing the cell concentration and enzyme productivity, fed-batch method was employed. However, in this case the cell growth rate was inhibited by accumulation of acetic acid. The cell growth rate decreases from 3.367 DCW/hr to 0.965 DCW/hr. When cell-recycle method was adopted, it was shown to decrease the accumulation of acetic acid. The cell growth rate increased to 2.62 DCW/hr with its cell concentration reaching 28.1 g-DCW/l. The enzyme volumetric activity rised from 4.76 IU/ml to 5.92 IU/ml.
|Appears in Collections:||化學工程學系所|
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