以全合成培養基饋料批次培養 Pichia pastoris 生產 Candida rugosa 脂肪脢三

Abstract

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.