利用 Pichia pastoris 生產 Candida rugosa 脂肪酶第三型

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 YPD 為基礎培養基，探討Candida rugosa 脂肪酶第三型 (CRL3) 之最適生產條件。三角瓶培養結果，菌體量 (OD600) 由 50 提升至 170，CRL3酵素活性由4.5 U ml-1 提升至180 U ml-1。培養基之起始 pH 值會影響菌體生長與酵素表現。利用五公升桌上型發酵槽之批次培養時， pH 9雖可得到較高濃度的菌體量及總蛋白表現量，但 CRL3 所佔的比例較低。控制於 pH 6 時可得最高酵素表現量，活性 140 U ml-1，酵素蛋白質 83.2 mg l-1，為 pH 8 時之六倍。最後進行饋料批次培養結果酵素活性提升至 210 U ml-1，酵素蛋白質為 103.48 mg l-1。

Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, was used to produce Candida rugosa lipase 3 (CRL3). Optimal conditions for the enzyme production were investigated in this study. Under the optimal conditions developed, biomass concentrations expressed as OD600 were increased from 50 to 170 and enzyme activities of CRL3 were increased from 4.5 U ml-1 to 180 U ml-1, respectively, after 5 days cultivation in shaking flasks and were shown to be influenced by initial pH of the medium. Using a 5-liter benchtop fermentor, production process of batch-type cultivation was further studied under pH controlled at 5, 6, 7 and 8. Increasing the pH of control resulted in increase of the amounts of biomass and total protein production. However amounts of CRL3 obtained were low. The enzyme activity of 140 U ml-1, with a 6-fold higher than that controlled at pH 8 was achieved under the condition of pH controlled at 6. The yield was 83.2 mg l-1 of growth media. The enzyme activity of 210 U ml-1 at fed-batch culture. The yield was 103.48 mg l-1 of growth media.