利用重組 Pichia pastoris 生產 Candida rugosa 脂肪酶 3 之生產菌株再改良與培養條件最適化

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能外泌 Candida rugosa 脂肪酶 3 (CRL3) 的 Pichia pastoris GS115/CRL3 菌株，首先進行菌株改良，以 Zeocin 作為篩選標記，逐步提升其濃度以篩選具較佳異源基因表現之轉形株。當 Zeocin 濃度為 1500 μg/mL 時，篩得具最高 CRL3 活性之菌株，命名為 Pichia pastoris GS115/CRL3/Z1500。利用 500 mL Hinton`s 搖瓶，採三因子-三水準 Box-Behnken 設計之反應曲面法 (Response surface methodology, RSM) 探討此新轉形株之最適培養條件，當 FM22 培養基碳氮比為 12、接種量 8.8% 及溫度15℃，震盪培養 120 小時之 CRL3 活性為 8.47 U/mL。以該條件於五公升醱酵槽內進行批式培養，控制 pH 5.0 條件下培養之酵素活性比未控制者佳，可達 26.77 U/mL。進一步進行饋料批式培養，以 11.25 mL/h 固定流速饋料培養 216 小時，菌體濃度 (OD600) 與酵素活性分別可達 627.6 與 451.7 U/mL，但培養液中有大量甘油累積；改採仿指數饋料模式結果，甘油累積現象可大幅改善，且培養 168 小時即可得到與固定流速饋料模式相當之酵素活性 (423.2 U/mL)，生產力可提高 20%，達 2.52 U/mL/h。

Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, was used to produce extracellular Candida rugosa lipase 3 (CRL3). Production strain was further improved by screening and selection on a series of agar plates in stepwise increase of Zeocin concentrations. The highest-yield strain was finally obtained and named as Pichia pastoris GS115/CRL3/Z1500. Using 500-mL Hinton`s flasks, response surface methodology (RSM) based on a three-factor three-level Box-Behnken design of experiment was used to optimize the culture conditions for CRL3 production by P. pastoris GS115/CRL3/Z1500. The optimal culture conditions were found as follows: C/N ratio in FM22, 12; inoculums size, 8.8%; temperature, 15℃. Under the optimal conditions, CRL3 activity of 8.47 U/mL was achieved after 120 h cultivation. Then, batch cultures were carried out in a 5-L bench-top fermentor under the optimal conditions. It was found that pH controlled at 5 was better than pH uncontrolled during cultivation. And a CRL3 activity of 26.77 U/mL was obtained after 96 h cultivation. Based on the result obtained, fed-batch cultures were further performed in order to attain the high cell density culture. While constant feeding rate at 11.25 mL/h was operated, OD600 of 627.6 and CRL3 activity of 451.7 U/mL were obtained, respectively after 216 h cultivation. However, a large amount of glycerol was accumulated in culture broth. While pseudo-exponential feeding mode was used instead, glycerol accumulation was minimized and CRL3 activity of 423.2 U/mL was achieved after 168 h cultivation. Productivity of 2.52 U/mL/h was obtained which was 20% higher than that of constant feeding mode.