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KMID : 0613820060160020274
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Journal of Life Science
2006 Volume.16 No. 2 p.274 ~ p.281
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Cloning and High Expression of Nattokinase Gene from Bacillus subtilis BB-1
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Lee Young-Hoon
Lee Sung-Ho
Park Ki-Hoon
Choi Young-Ju
Jeong Yong-Kee
Gal Sang-Wan
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Abstract
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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℃, 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.
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KEYWORD
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Nattokinase, fibrinolytic enzyme, Bacillus-E. coli shuttle vector, polymerase chain reaction
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