利用實驗設計法探討重組幾丁質酵素生產之最佳化

Optimization of Recombinant Chitinase Production by Experimental Design Methods

林宗翰(Tsung-Han Lin)

指導教授 ： 莊瑞鑫

元智大學/工程學院/化學工程與材料科學學系/碩士(2011年)

摘要

近年來，多數的重組蛋白質在工業、農業或是生物醫學上之應用急遽增加，而利用Escherichia coli生產這些重組蛋白質是一個既快速且經濟的方式，故本研究以E. coli BL21 (DE3) 及E. coli DH5α作為表達重組蛋白質His-tag及GST之菌株，進行探討生產重組幾丁質酵素 (ASChi61) 之活性，其分子量為61 kDa。並以cobalt resin及GSH agarose分別對其進行純化，實驗結果發現，重組幾丁質酵素於宿主細胞E. coli BL21 (DE3) 之表現較E. coli DH5α佳。
以醱酵槽進行E. coli BL21 (DE3)/pET22b-CTS61之培養，並利用實驗設計法探討誘導時之pH值、溫度、轉速、濃度以及時間對重組幾丁質酵素之影響，從部分因子效應分析結果可判斷出，溫度和時間之交互影響效應較為顯著，利用反應曲面法進一步探討最適操作條件，結果顯示當溫度為23.9℃及誘導時間為115 min時，可得預測之重組幾丁質酵素總活性為32,120 U，利用反應曲面法所得之最適操作條件，進行醱酵槽實驗測試，其重組幾丁質酵素平均總活性為30,650 U，相較於預測值其誤差為4.8 %，而經最適化後之重組幾丁質酵素總活性提升了1.54倍。

關鍵字

幾丁質酵素 ； 基因重組大腸桿菌 ； 實驗設計法

並列摘要

Chitin and its derivates are multi-functional molecules. The hexameric saccharides pose potent biomedical function. In the past few years, hexaoligochitin produced by chitinase, ASChi61, from Aeromonas schubertii was identified. The enzyme is an SDS-resistant and was constructed in expression vectors.
In recent years, numerous recombinant proteins used for industrial, agricultural or biomedical applications have increased dramatically. Mass production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of these proteins. In this work, the expression systems, pET22b vector in E. coli BL21 (DE3) and pGEX2T vector in the E. coli DH5α for producing recombinant protein ASChi61 were evaluated. After purification, more recombinant ASChi61 was obtained in pET22b vector with E. coli BL21 (DE3).
Then the efficiency of recombinant protein expression was studied by various parameters. Interactions between these parameters often increase the complexity of identifying those which are the key to improve protein expression. The optimization experiments were simplified by statistical design of experiments. E. coli BL21 (DE3)/pET22b-CTS61 was cultivated in the fermentator with the method of experimental design, during the induction of pH, temperature, stirring rate, inducer concentration and time on the recombinant protein. From the fractional factorial design, the effect of temperature and time interaction effect were more significant. The optimum operating conditions was explored by response surface methodology. It predicited the total activity of the enzymes was 32,120 U at 23.9℃ with 115 min of induction. Under that conditions, the total activity of recombinant protein was 30,650U in fermentation experiment. The error is only 4.8%. And the total activity of ASChi61 increased 1.54-fold in the optimal operating conditions.

並列關鍵字

chtinase ； recombinant protein ； design of experiment

參考文獻

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