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標題: | 以全合成培養基饋料批次培養 Pichia pastoris 生產 Candida rugosa 脂肪脢三 Production of Candida rugosa lipase 3 by Pichia pastoris using fed-batch cultivation in synthetic medium |
作者: | Chung-Yu Yu 余忠佑 |
指導教授: | 黃健雄(Jan-Hsiung Huang) |
關鍵字: | 饋料批次培養,CRL3,Pichia pastoris, CRL3,fed-batch cultivation,Pichia pastoris, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 使用中央研究院植物暨微生物學研究所蕭介夫教授研究室所構築之 Pichia pastoris GS115/CRL3,以前人研究結果 (侯,2006;李,2007) 為基礎,進一步探討 Candida rugosa 脂肪酶三 (CRL3) 於五公升桌上型醱酵槽大量表現之最適條件。以修改過之 YPG 複合培養基在 700 rpm、25oC、pH 6 下批次培養至 48 小時,培養液之菌體濃度 (OD600 nm) 152,CRL3 活性 84 U mL-1,蛋白酶活性 0.22 U mL-1。使用 FM22 全合成培養基在相同條件下,培養液之 OD600 nm 78,CRL3 活性 23 U mL-1,細胞體積變小,但測不出蛋白脢活性,也發現菌體生長與 histidine 添加量有關。以補充 3 g L-1 histidine 之 FM22 為起始培養基,含 2 g L-1 histidine 之 50% 甘油為饋料液,當起始培養基中甘油用盡時開始以固定速率添加饋料液。在 1000 rpm、25oC、pH 5.0、饋料速率 16.6 mL h-1,饋料批次培養 192 小時培養液之細胞乾重 123.4 g L-1 (OD600 nm 590),CRL3 活性 342.8 U mL-1,較前人 (侯,2006) 於複合培養基饋料批次培養之結果,菌體量增加 3 倍,酵素活性增加 0.7 倍。 Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, is used to produce Candida rugosa lipase 3 (CRL3). Batch cultivation was demonstrated in a 5-L bench-top fermentor containing complex medium of YPG modified by our previous studies. After 48 h cultivation, cell density of OD600 nm 152, CRL3 activity of 84 U mL-1 and protease activity of 0.22 U mL-1 were obtained under culture conditions of 700 rpm, 25oC and pH 6.0. While a synthetic medium of FM22 was used, cell densities of OD600 nm 78 and CRL3 activity of 23 U mL-1 were obtained under the same conditions. Histidine requirement for cell growth and smaller cells with no protease activity were also found. A fed-batch fermentation process was then developed using FM22 supplemented with 3 g L-1 histidine as an initial medium. At the depletion of the batch glycerol, fed-batch phase was started by feeding a 50% glycerol solution containing 2 g L-1 histidine at a constant feeding rate of 16.6 mL h-1. After 192 h cultivation, cell density of 123.4 g L-1 DCW (OD600 nm 590) and CRL3 activity of 342.8 U mL-1 were achieved under 1000 rpm, 25oC, and pH 5.0. The yield of biomass and CRL3 activity were improved 3-fold and 0.7-fold, respectively, compared with our previous studies using complex medium in a fed-batch strategy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24856 |
全文授權: | 未授權 |
顯示於系所單位: | 微生物學科所 |
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