利用複合培養基與甘蔗糖蜜以 Pichia pastoris 搖瓶培養生產Candida rugosa 脂肪

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 (侯，2006) 最適化之培養條件為基礎，進一步探討Candida rugosa 脂肪酶三 (CRL3) 之最佳醱酵生產條件。在Hinton 氏瓶的探討中，於前人最適化之YPG 培養基中額外添加2 % beef extract 時，菌體濃度與酵素活性均上升，其中酵素活性達到255 U/mL，較前人搖瓶探討所得之最佳活性 (180 U/mL) 提升42 %，產量為110.2 mg/L；當再添加1.5 % 橄欖油，酵素活性再提升至295 U/mL。添加1 - 5 mM PMSF (Phenylmethylsulphonyl fluoride)、1 -5 μg/mL Pepstatin A 與 1 - 5 mM EDTA (Ethylenediaminetetraacetic acid) 等蛋白 酶抑制劑均無法有效提升酵素活性。粗酵素液在中性環境有較佳活性，且較適合保存在中性與30 oC 以下的環境中。為了降低生產成本，使用製糖副產物甘蔗糖蜜進行CRL3 生產。於7 % (以蔗糖濃度計) 甘蔗糖蜜、 0.2 % 尿素、0.15 % (v/v)之 85 % 磷酸與1.5 % (w/v) 玉米浸漬液之培養基中，25 oC、125 rpm 下，以Hinton 氏瓶震盪培養120 小時，OD600 為69.3，酵素活性為45.4 U/mL。

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). Based on the cultivation condition optimized by Hou (2006) in Hinton’s flask, the activity of CRL3 increases from 180 to 255 U/mL when 2 % beef extract is added as extra nitrogen source and yield of CRL3 is 110.2 mg/L, a 42 % increase over previous study. Furthermore, in the presence of 1.5 % olive oil, the activity reaches 295 U/mL. However, addition of protease inhibitors in the range of 1-5 mM PMSF, 1-5 μg/mL pepstatin A and 1-5 mM EDTA, respectively, do not improve the enzyme expression. Crude CRL3 has an optimal reaction and storage conditions at pH 7.0 and temperature below 30 oC. While a low-cost medium, composed of 7 % cane molasses (as sucrose), 0.2 % urea, 0.15 % (v/v) 85 % phosphoric acid and 1.5 % corn steep liquor, was used, OD600 of 69.3 and CRL3 activity of 45.4 U/mL were achieved in Hinton flasks at 25 oC, 125 rpm for 120 hours.