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題 名 | 甲醇指數進料策略對於重組Pichia pastoris生產豬表皮生長因子之影響=Effect of Methanol Exponential Feeding Strategy on Production of Recombinant Porcine Epidermal Growth Factor from Pichia Pastoris |
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作 者 | 洪玉祥; 余秉樺; 張永鍾; 陳博彥; 李德南; | 書刊名 | 宜蘭大學生物資源學刊 |
卷 期 | 3:1 民95.12 |
頁 次 | 頁53-59 |
分類號 | 368.5 |
關鍵詞 | 甲醇指數饋料速率; 豬表皮生長因子; Methanol exponential feeding rate; Pichia pastoris; Porcine epidermal growth factor; |
語 文 | 中文(Chinese) |
中文摘要 | 以生長相後接基因表達生產相之二相式饋料批次培養系統,以甘油當作生長相之碳源基質,而於生產相則以甲醇作為碳源及生產重組蛋白質之誘發劑,評估不同甲醇進料速率對甲醇快速利用型Pichia pastoris菌株(GS115)之過程,以比較不同甲醇指數饋料速率(μ□)對細胞生長動力學和豬表皮生長因子(pEGF)生產量之影響。發酵過程先經歷24 h以上之甘油饋料期,甘油之饋料速率設定為29.3 ml h¯¹,當濕細胞約重達150g l¯¹,即進行甲醇指數饋料期。甲醇指數饋料期間除以甲醇取代甘油外,其餘發酵條件皆與甘油饋料期相同。μ□設定為0.010、0.026、0.030、0.035和0.050 h¯¹,發酵期間間隔1-12 h收集樣品進行記錄發酵液總量、細胞重、pEGF濃度及甲醇濃度。結果顯示,不同μ□處理之濕細胞重與pEGF濃度皆隨發酵時間增加而提高,當μ□=0.030 h¯¹時在進行甲醇饋料後48 h有較高的濕細胞重,而μ□=0.030-0.035 h¯¹在進行甲醇饋料後36 h即有較高的pEGF濃度,而μ□=0.050 h¯¹時,其在甲醇饋料36 h後即會開始累積甲醇。由以上試驗結果發現細胞生長與pEGF生產量間呈現正相關性,當μ□=0.028-0.029 h¯¹時有最佳pEGF生產量,當與0.010 h¯¹比較時,其pEGF生產速率約可從5.8增加到12.4 mg/l‧h。 |
英文摘要 | This study provided an exponential feeding strategy in two-phase fed-batch cultivation of a methanol-utilizing plus (Mut[90a7]) pichia pastoris strain. To optimize foreign gene expression and decrease the metabolic burden to cell growth, glycerol and methanol were used as sole carbon sources in the first and second phase to genetically enhance the growth phase and production phase, respectively. In particular, methanol not only played a role as a carbon substrate, but also an inducer for target protein expression. Prior to genetic induction, glycerol-containing cultures were firstly carried out for approximately 24 hours (feeding rate set at 29.3ml h¯¹). Once the wet cell concentration was achieved to 150g 1¯¹, the feeding substrate was switched to methanol in an exponential feeding strategy to induce pEGF production (all other conditions remained identical). The optimal exponential rate for methanol feeding was determined (μ□) to maximize the production of porcine epidermal growth factor (pEGF) using a Mut[90a7] pichia pastoris strain (GS115) a model system. The growth behavior and production characteristics at μ□ of 0.010, 0.026, 0.030, 0.035, and 0.050 h¯¹ clearly revealed a significant correlation between cell growth and pEGF production, indicating the nature of growth-associated production. In addition, the optimal pEGF production approximately occurred at 0.028 – 0.029 h¯¹. Compared to theμ□ at 0.010 h¯¹, this optimal pEGF productivity increased from 5.8 to 12.4 mg/1.h. |
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