以大腸桿菌異源表現納豆激

HETEROLOGOUS EXPRESSION OF THE NATTOKINASE GENE IN ESCHERICHIA COLI

林峰毅(Feng-Yi Lin)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

納豆激是由Bacillus subtilis natto所分泌之胞外蛋白，其具有優異的分解血栓能力。已知在腸道內可以被快速吸收且作用時間長，因此在保健食品和藥品上的應用格外受到注目。
本研究將納豆激的對應基因建構於大腸桿菌質體pET26b(+)上進行異源表現，並藉由sorbitol和betaine於培養液中的添加，成功表現產生具有溶解血栓能力的重組納豆激。此外，藉由培養液組成、培養溫度、誘導時菌體濃度的調整，我們使大腸桿菌異源表現納豆激的能力進一步提升。以添加sorbitol和betaine的TB培養，於菌體濃度OD600 = 1.5時以IPTG誘導，並將表現培養溫度調整為25℃，可於誘導後4小時，測得較佳胞內酵素活性約1.66 U/ml。而胞外酵素活性，則於菌體濃度OD600 = 1.2時，在25℃下誘導培養10小時後，可得較佳活性約6.1 U/ml。根據重組納豆激之casinolytic activity的檢測，最佳反應溫度為50℃，而最佳反應pH為10。

關鍵字

納豆激 ； 異源表現 ； 納豆

並列摘要

Nattokinase is an extracellular protease made by Bacillus subtilis natto which is isolated form Natto, has a excellent ability for digesting fibrin. It can be absorbed quickly by intestine, not only created a strong fibrinolytic activity in human body, but maintained the effect more then eight hours.
In this study, we cloned nattokinase’s gene－aprN at pET26b(+) and transformed into Escherichia coli expressing system. Then we successes expressing recombinant nattokinase which has the fibrinolytic activity by added sorbitol and betaine in medium. Besides, we improved the fibrinolytic ability of nattokinase by adjusting medium consist、changing incubated temperature and inducing protein expression at different bacteria numbers.
Using TB (contained sorbitol and betaine) incubated recombinant E. coli until OD600=1.2, adjusting temperature form 37℃ to 25℃,the higher endocellular enzyme activity of 1.66 U/ml was obtained 4 hours after induction. On the other hand, the higher excellular enzyme activity of 6.1 U/ml was obtained 10 hours after induction at the same condition.

並列關鍵字

nattokinase ； heterologous ； expression ； natto

參考文獻

Berliner J.A., and J.W. Heinecke. 1996. The role of oxidized lipoproteins in atherogenesis. Free radical biology and medicine. 20(5):707-27

Cai Q., and H. Wei. 1996. Effect of dietary genistein on antioxidant enzyme activities in SENCAR mice. Nutrition and Cancer. 25(1):1-7.

Chang C.T., M.H. Fan, F.C. Kuo, and H.Y. Sung, 2000. Potent fibrinolytic Enzyme from a mutant of Bacillus subtilis IMR-NK1. Journal of agricultural and food chemistry. 48: 3210–3216.

Chiang C.J., H.C. Chen, Y.P. Chao, and J.T. Tzen. 2005. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli. Journal of agricultural and food chemistry. 53(12):4799-804.

Choi N.S., K.T. Chang, P.J. Maeng, and Kim S.H. 2004. Cloning, expression, and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4. FEMS microbiology letters. 236 (2) :325-31.