透明顫菌血紅蛋白共表現對於納豆激酶於大腸桿菌異源表現的影響

Effect of Vitreoscilla hemoglobin co-expression on heterologous expression of nattokinase in E. coli

黃佩萱(Pei-Hsuan Huang)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2007年)

摘要

由於近年來心血管保健的議題受到高度重視，具有強效分解血栓功能的納豆激酶遂成為熱門的研究對象。本研究將細菌血紅蛋白(Vitreoscilla hemoglobin)基因，插入先前建構之含納豆激酶基因的E. coli質體中，欲藉由細菌血紅蛋白的表現，幫助菌體生長並提高目標蛋白表現量。另ㄧ方面，亦藉由培養條件如誘導溫度和起始誘導菌體濃度的調整，進一步提升異源表現納豆激酶的能力。以添加sorbitol和betaine的TB為培養液，於菌體濃度OD600=1.8時誘導，並將培養溫度調降為20℃，可於誘導後8小時測得較佳胞內酵素活性8.7 U/ml。利用metal chelating affinity純化重組納豆激酶，求得比活性為406.4 U/mg。純化重組納豆激酶在50℃下有最好活性， Tm值約47℃，pH 10呈現最高相對活性，而在pH 9下具有最佳的穩定度。

關鍵字

納豆激酶 ； 透明顫菌血紅蛋白 ； 大腸桿菌

並列摘要

In recent years, there has been an increasing attention on the cardiovascular health. Nattokinase, a highly fibrinolytic enzyme, has become the focus of study. In this study, the Vitreoscilla hemoglobin (VHb) gene was inserted into a previously constructed nattokinase gene- containing E. coli recombinant plasmid. The expression of VHb was intended to help the growth of bacteria and promote the expression level of target protein. The expression temperature and bacterial cell concentration at induction was adjusted to further facilitate the enzyme expression. With sorbitol and betaine-containing terrificbroth as the culture medium, the bacterial culture was induced after OD600=1.8, and the expression temperature was shifted from 37oC to 20 oC. The maximal intracellular nattokinase activity obtained was 8.7 U/ml, which occurred 8 hours after induction. The specific activity of purified nattokinase was 406.4 U/mg. The optimal temperature for nattokinase activity and Tm were 50 and 47℃, respectively. The optimal pH for nattokinase activity was 10, while the enzyme was most stable at pH 9.

並列關鍵字

E. coli ； nattokinase ； VHb

參考文獻

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