使用不同質體和透明顫菌血紅素異源共表現納豆激酶於大腸桿菌中

The heterologous expression of nattokinase using different plasmids and its coexpression with Vitreoscilla hemoglobin in Escherichia coli

施淑觀(Shu-kuan Shih)

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

本研究以S10啟動子驅動Vitreoscilla hemoglobin (VHb)和經基因融合的double VHb基因，並與納豆激酶共同建構於pET26b上，在Escherichia coli中進行異源表現。以含betaine與sorbitol的TB做為培養基時，兩者皆約在IPTG誘導後4小時達到最高胞內活性約2.5 U/ml，胞外活性則在誘導後20小時達高峰，約2.6 U/ml。相較於無VHb共表現的E. coli，活性約增加27 %，且活性表現持續時間較久。與活性高峰相比，於誘導後20小時，有VHb表現之E. coli仍具60%活性；反之，無VHb表現之E. coli已無活性。在蛋白質純化的部分，得到pProNAT、pVN和pdVN之比活性分別為384、402和440 U/mg。另方面嘗試將納豆激酶基因架構於pET20b、pET23am、pET24a，和Bacillus subtilis質體－pDual374；但活性表現皆不如建構於pET26b上的，最高胞內活性依次僅約1.1、0.88、0.7、0.028及0.025 U/ml。

關鍵字

血紅素 ； 納豆激酶 ； 透明顫菌

並列摘要

In this study, the Vitreoscilla hemoglobin (VHb) gene and the double VHb gene (dVHb) derived from the fusion of two single VHb genes were coexpressed with nattokinase in Escherichia coli. In the presence of VHb or dVHb and using TB containing betaine and sorbitol as culture medium, the maximal intra- and extracellular activities were about 2.5 and 2.6 U/ml, respectively, which were obtained 4 and 20 h, respectively, after induction with IPTG. The nattokinase activity resulted from coexpression with VHb was increased by 27 % in comparison with that expressed alone. The specific activities of pProNAT、pVN and pdVN are 384、402 and 440 U/mg, respectively.The nattokianse gene was also inserted into pET20b, pET23am, pET24a and pDual374 (a Bacillus subtilis plasmid). However, all the expressions from these recombinant plasmids were lower than that from the recombinant pET26b with the corresponding activities of 1.1、0.88、0.7 、0.028 and 0.025 U/ml, respectively.

並列關鍵字

nattokinase ； Vitreoscilla ； hemoglobin

參考文獻

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