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http://dx.doi.org/10.5352/JLS.2006.16.2.274

Cloning and High Expression of Nattokinase Gene from Bacillus subtilis BB-1  

Lee Young-Hoon (Department of Microbiological Engineering, Jinju National University)
Lee Sung-Ho (Department of Biology, Gyeongsang Natinal University)
Park Ki-Hoon (Department of Applied Life Science, Gyeongsang National University)
Choi Young-Ju (Department of Food and Nutrition, Silla University)
Jeong Yong-Kee (Department of Life science and Biotechnology, Dong Eui University)
Gal Sang-Wan (Department of Microbiological Engineering, Jinju National University)
Publication Information
Journal of Life Science / v.16, no.2, 2006 , pp. 274-281 More about this Journal
Abstract
A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.
Keywords
Nattokinase; fibrinolytic enzyme; Bacillus-E. coli shuttle vector; polymerase chain reaction;
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