Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli.
Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.
Liang
Xiaobo
X
State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beijing, China.
Jia
Shifang
S
Sun
Yufang
Y
Chen
Meiling
M
Chen
Xiuzhu
X
Zhong
Jin
J
Huan
Liandong
L
D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D011480:Protease Inhibitors / Q000494:pharmacology; D022142:Secretory Vesicles / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D011480:Protease Inhibitors; D013381:Subtilisins; C053771:nattokinase