Molecular Cloning and Expression of Nattokinase Gene in Bacillus subtilis

LIU Bei-Yu, SONG Hou-Yan^*
( Department of Molecular Genetics, School of Medicine,Fudan University,Shanghai 200032,China )

Abstract    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR.The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene.The supernatant of the culture was collected after 15 h culture.The target proteins were identified by SDS-PAGE.Nattokinase was purified by a method including ultrafiltration,Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography,and 100 mg of purified nattokinase was obtained from one liter of culture.The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively.
Key words    nattokinase; cloning and expression; purification; Bacillus subtilis

^*Correspondering author: Tel, 86-21-64041900-2092; Fax, 86-21-64033738; e-mail, [email protected]