探討轉殖大腸桿菌生產外切型纖維分解酵素 之最適培養條件

Investigation of optimal culture conditions for the production of 1,4-exoglucanase by recombinant Escherichia coli

陳怡綺(Yi-chi Chen)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2011年)

摘要

纖維素分解酵素(cellulase)廣泛分佈於昆蟲、軟體動物、原生動物及微生物群。微生物以真菌為最多，此外高溫性放線菌、藻類、酵母、黏菌等也可分泌纖維素分解酵素。本篇研究為採用不同醱酵策略，以基因轉殖E. coli生產外切型纖維分解酵素。由實驗結果發現，以LB(Luria Bertani)為初始培養基，饋入五碳糖/酵母萃取物/胰蛋白胨 (0.5/1/0.5, w/w)，則得活性為693 U/L，蛋白質濃度為944 mg/L。若饋入五碳糖/酵母萃取物 (1/1, w/w)，可得最高活性為1467 U/L，蛋白質濃度為4239 mg/L，証實tryptone不利於活性之提升。若以醇類為基礎之TB培養基，採用饋料為五碳糖/酵母萃取物 (1/1, w/w)進行酵素誘導，則最高活性為1469 U/L，最高蛋白質濃度為4793 mg/L。於誘導溫度實驗發現，於低溫下誘導活性為1477 U/L，而於常溫下誘導之活性為1231 U/L，證明若考慮能源之損耗，可以適當提升其誘導溫度。而使用不同種類誘導劑，如0.5 mM IPTG，0.02 mM Lactose，0.2 mM Lactose，與2 mM Lactose，則最高活性為1888 U/L，證明可以較便宜之Lactose取代IPTG來減少生產成本。於分析批次醱酵中醇類濃度對於菌體生長之影響，以數學模式獲得μmax為0.80 g/L•h，Ks則為0.43 g/L，此數值可運用於模擬不同饋料策略，以探討短時間內大量生產外切型纖維分解酵素之目標。

關鍵字

饋料醱酵 ； 外切型纖維分解酵素 ； 大腸桿菌

並列摘要

Cellulase is widely distributed in insects, molluscs, protozoa and microorganisms. Most fungi, in addition to thermophilic actinomycetes, algae, yeast, and sticky monocytogenes can secret cellulase. Different fermentation strategies were investigated in this study for the production of 1,4-exoglucanase from the recombinant E. coli. The results showed that by using LB(Luria Bertani) as the initial medium, feeding G/YE/Try as ratio of 0.5/1/0.5 w/w, resulted in the volumetric activity of 693 U/L, and protein yield of 944 mg/L. Feeding G/YE as ratio of 1/1 w/w without tryptone, could get the higher volumetric activity of 1467 U/L, and protein yield of 4239 mg/L. Therefore it confirmed that tryptone had the inhibitory on the volumetric activity.
　　By using TB as a alcohols-based medium, feeding G/YE as ratio of 1/1 w/w for the 1,4-exoglucanase induction resulted in the highest volumetric activity as 1469 U/L, maximum protein yield of 4793 mg/L. To study the induction temperature effect on the volumetric activity, it shown that lower temperature resulted in higher activity of 1477 U/L, but higher temperature resulted in slightly lower activity of 1231 U/L. Therefore, for the purpose of energy-saving, we could use the room temperature for the induction of enzyme production. By studying different inducing agents as 0.5mM IPTG, 0.02 mM Lactose, 0.2 mM Lactose,and 2 mM Lactose resulted in the highest volumetric activity as 1888 U/L, therefore, we could use cheaper lactose to replace IPTG. On the analysis of alcohols effects on the cell growth, the Monod model shown the μmax as 0.80 1/h, and Ks as 0.43 g/L. The kinetic model can be applied to simulate different feeding strategies for the mass production of cellulase in a way.

並列關鍵字

fed-batch fermentation ； exoglucanase ； E. coli

參考文獻

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