不同誘導劑對大腸桿菌異源表現納豆激酶之影響

EFFECT OF DIFFERENT INDUCERS ON THE HETEROLOGOUS EXPRESSION OF THE NATTOKINASE GENE IN ESCHERICHIA COLI

郭莉婷(Li-Ting Kuo)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2009年)

摘要

納豆激酶是由Bacillus subtilis natto所分泌之胞外蛋白酶，具有優異的分解血栓能力。目前已知在腸道內可以被快速吸收且作用時間長達8小時以上，因此在保健醫藥品的應用上格外受到注目。本研究將建構於大腸桿菌質體pET26b(+)的納豆激酶基因，藉由於TB中添加sorbitol和betaine，成功表現產生具有溶解血栓能力的重組納豆激酶。單獨以IPTG誘導時(最佳誘導濃度為1 mM IPTG)，可於誘導後24小時，測得較佳胞外酵素活性約2.71 U/ml;誘導後8小時，測得較佳胞內酵素活性約4.67 U/ml。以lactose誘導(最佳誘導濃度為10 mM)時，可於誘導後16小時，測得較佳胞外酵素活性約1.29 U/ml;誘導後8小時，測得較佳胞內酵素活性約1.75 U/ml。IPTG與lactose共同誘導時，最佳誘導濃度為10 mM lactose和0.125 mM IPTG，可於誘導後20小時，測得較佳胞外酵素活性約3.36 U/ml;誘導後4小時，測得較佳胞內酵素活性約3.5 U/ml。

關鍵字

納豆激酶

並列摘要

Nattokinase, an extracellular protease produced by Bacillus subtilis natto exhibits excellent fibrinolytic activity. It can be absorbed quickly by intestine and has prolonged effect over eight hours. Thus, nattokinase has raised great attention for the potential applications in healthy protection and medicine. In this study, the nattokinase gene constructed on pET26b(+) was successfully expressed in TB containing betaine and sorbitol to produce nattokinase with fibrinolytic activity.

Using IPTG as the inducer solely, the maximal intracellular nattokinase activity of 4.67 U/ml was obtained 8 hours after induction by 1 mM IPTG, while the maximal extracellular activity of 2.71 U/ml was obtained 24 hours after induction by the same condition. As lactose was used as inducer instead, the higher intracellular activity of 1.75 U/ml was obtained 8 hours after induction by 10 mM lactose. Meanwhile, the higher extracellular enzyme activity of 1.29 U/ml was obtained 16 hours after induction. With lactose and IPTG as inducers after induction, the maixmal intracellular activity of 3.50 U/ml was obtained 4 hours and the maximal extracellular activity of 3.36 U/ml was detected 20 hours after induction by 10 mM lactose and 0.125 mM IPTG.

並列關鍵字

nattokinase

參考文獻

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