- 學位論文
以乳糖誘導基因重組大腸桿菌生產β-葡萄糖苷酶之醱酵策略探討
Strategies for Producing β-Glucosidase from Recombinant E. coli by Lactose-induced Fermentation
摘要
β-葡萄糖苷酶可分解纖維二醣及纖維素末端，釋放出葡萄糖分子。在纖維素被酵素分解的過程中，纖維二醣會抑制某些纖維素酶的活性，諸如: cellobiohydrolase (EC 3.2.1.91) 和endo-1,4-β-D-glucanase (EC 3.2.1.4)的活性。因此 β-葡萄糖苷酶在生物工程上是很重要的酵素。 本研究主要是利用乳糖誘導基因重組大腸桿菌生產一個源自高溫細菌的葡萄糖苷酶，首先將其基因建構在大腸桿菌 BL-21(DE3) 的質體 pET-21a中進行異源蛋白表現；為了降低成本，用乳糖取代 IPTG誘導，而且醱酵及誘導過程維持在25°C進行，不用冷凍機降溫。 生產基因重組β-葡萄糖苷酶的醱酵過程分為兩個主要階段: 前培養及誘導兩個階段。前培養階段主要以五公升醱酵槽採限制葡萄糖策略進行醱酵試驗，定時饋入葡萄糖和酵母萃取物（重量比為1：1）的混合物，使培養液的葡萄糖濃度維持在1g/L。醱酵條件為：操作體積3公升、溫度25°C、轉速300 rpm、通氣1 vvm，pH值控制在7.1-7.4。而誘導階段則是自五公升醱酵槽中取出100毫升醱酵液置於250mL的搖瓶中，添加誘導劑，以 25°C、150 rpm 培養至64小時。 探討不同培養基、誘導劑濃度、醱酵時間及誘導時間對生產β-葡萄糖苷酶的影響。以TB培養基加入十倍磷酸二氫鉀進行醱酵 12 小時，添加 8 mM 乳糖進行誘導後，繼續醱酵 64 小時可得最高的酵素活性，達 IV 到 74.1 U / mL。在一連串的實驗過程，培養基成分由LB改成添加磷酸鹽的TB，β-葡萄糖苷酶的活性由8.0 U/mL提高至74.1 U/mL，增加9.2倍。
並列摘要
β-Glucosidase hydrolyzes cellobiose and terminal residue of cellulose with release of glucose. Cellobiose can inhibit some cellulases, such as cellobiohydrolase (EC 3.2.1.91) and endo-1,4-β-D-glucanase (EC 3.2.1.4). Therefore β-glucosidase is an important enzyme in biotechnology. This work was engaged in the production of β-glucosidase originated from a thermophilic bacterium by culture of recombinant E. coli. The related gene had been constructed in pET-21a and was expressed by E. coli BL-21 (DE3). In order to reduce the cost, the inducer IPTG was replaced by lactose and the culture was maintained at 25°C, without decreasing temperature by refrigeration. For production of recombinant β-glucosidase, the fermentation process falls into two stages: preculture and induction. Preculture was carried out in a 5-L fermenter with a working volume of 3 L, at 25°C, 300 rpm, 1 vvm and pH being controlled in a range of 7.1-7.4. The induction process was performed by aseptically transferring 100 mL preculture into a 250-mL flask. After addition of IPTG or lactose as inducer, the flask culture was incubated in a 25°C shaker at 150 rpm for 64 h. In order to enhance the production of II recombinant β-glucosidase, the fermentation was carried out by varying the composition of the culture medium, the concentration of inducer, the preculture time and induction time. During the preculture stage, a glucose-limited fermentation was performed, wherein a mixture of glucose and yeast extract, with a mass ratio of 1:1, was periodically added to the culture broth to sustain glucose concentration at 1 g/L. An optimal β-glucosidase activity at 74.1 U/mL was obtained by E. coli cultured in a ten-fold potassium dihydrogen phosphate-reinforced TB medium after a 12-h preculture and an followed 64-h induction by adding 8mM lactose as inducer. As the culture medium was varied from LB to phosphate-reinforced TB, β-glucosidase activities increased from 8.0 to 74.1 U/mL, by a factor of 9.2.