利用乳糖誘導基因重組大腸桿菌生產β-葡萄糖苷酶之研究

Investigation of Lactose-Induced Production of β-glucosidase from Recombinant Escherichia coli

詹渝斐(Yu-fei Chan)

指導教授 ： 許垤棋

大同大學/工程學院/生物工程學系所/碩士(2012年)

摘要

β-葡萄糖是一種纖維分解酵素。纖維素的組成由葡萄糖單位藉由 β-1.4 glucosidation 結合而成的線狀聚合物。酵素分解纖維素的過程，首先由內切型纖維素分解酵素 (endo-β-1,4-glucanase) 將纖維素鍊切成較短的單位，纖維二糖水解酶 (cellobiohydrolase) 則從還原端切下纖維二糖，最後β-葡萄糖苷酶將纖維二糖分解為葡萄糖分子。一般微生物無法直接利用纖維二糖提供能量的來源。
本篇主要是利用饋料批次醱酵來進行基因重組大腸桿菌生產β-葡萄糖苷酶的研究，β-葡萄糖苷酶的基因來自耐高溫厭氧菌，轉殖在pET21-a質體上，利用乳糖和異丙基-β-D-硫代半乳糖苷 (IPTG) 去做基因表現。
細菌的前培養使用醱酵槽操作總體積為3公升、溫度25°C、轉速500 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 mL醱酵液置於250 mL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Luria-Bertain（LB）培養基進行簡單的半批式醱酵培養實驗結果在醱酵第6小時添加8 mM乳糖進行誘導後，繼續醱酵64小時的活性為17.89 U/mL。以添加磷酸鹽的Terrific Broth (TB) 培養基進行簡單的半批式 (semi-batch) 醱酵培養可獲得最佳活性。在醱酵 8 小時添加 8 mM 乳糖進行誘導後，繼續醱酵 64 小時可得最佳的酵素活性，達到 82.04 U / mL。比較LB與TB的實驗結果，後者酵素活性比前者提高4.59倍。

關鍵字

乳糖 ； β-葡萄糖苷酶 ； 基因重組大腸桿菌

並列摘要

β-Glucosidase is a cellulolytic enzyme. Cellulose is composed of glucose units by β-1.4 glucosidation with a combination of linear polymer chains. Enzymatic decomposition of cellulose is initialized with endo-cellulase (endo-β-1 ,4-glucanase) which cuts cellulose chains into shorter units. Cellulohydrase hydrolyzes cellulose into small glucosidic molecules with reducing ends, such as cellobiose. In the final step, β-glucosidase cuts cellobiose into glucose molecules. Generally microorganisms can not directly get energy by consuming cellobiose.
The present work is engaged in the production of β-glucosidase by fed-batch fermentation of a recombinant E.coli. β-Glucosidase encoded gene from a thermophilic anaerobe was constructed in pET21-a plasmid. Not only isopropyl-β-D-thiogalactoside (IPTG) but also Lactose were used for the gene expression. Preculture was carried out using a 5-L fermenter with a working volume of 3 L, at 25℃, 300 rpm, 1 vvm and pH being controlled in a range of 7.1-7.4. The induction of β-glucosidase was carried out by transferring 100 mL of preculture into a 250-mL flask aseptically. After addition of different inducer, the flask was incubated in a 25℃shaker at 150 rpm for 64 h.
The fermentation was carried out by varying the composition of the culture medium composition, the concentration of inducer, the preculture time and induction time. When Luria Bertani (LB) was used as the culture medium, a maximum β-glucosidase activity at 17.89 U/mL was obtained after a 6-h preculture and a 64-h induction by adding 8 mM lactose as inducer.
When Terrific Broth (TB) was used as the culture medium, the highest β-glucosidase activity up to 82.04 U/mL was obtained after a 8-h preculture and a 64-h induction by adding 8 mM lactose as inducer. β-Glucosidase activity obtained using phosphate-reinforced TB as culture medium was 4.59-fold higher than that LB was used.

並列關鍵字

Lactose ； β-Glucosidase ； recombinant Escherichia coli

參考文獻

1.楊久盈。2011。以乳糖誘導基因重組大腸桿菌生產β-葡萄糖之醱酵策略探討。大同大學生物工程研究所碩士論文。

2.蔡怡韋。2008。葡萄糖穩態控制醱酵對於基因重組E.coli細胞調控與Colicin E7 Nuclease之生產研究。大同大學生物工程研究所碩士論文。

5.袁秀榕。2010。本土多黏類芽孢桿菌Bs 83 β-葡萄糖之分離純化與特性分析。朝陽科技大學生化科技研究所碩士論文。

3.柯博順。2004。以大腸桿菌生產重組蛋白之醱酵策略探討。國立成功大化學工程研究所碩士論文。

6.Auger EA, Bennett GN, 1987, Temperature optimization of in vivo expression from the E. coli trp and trp:lac promoters. Biotechnol Lett 8:157-162.