An Experimental Study On Expressing Nattokinase Gene In Saccharomyces Cerevisiae

Nattokinase (NK) is a kind of fibrinolytic enzyme that is extracted from natto - a traditional food in Japan. Compared with other thrombolytic drugs which are used in common, NK is safer and easier to be absorbed by body, and has more direct and more persistent effect on thrombosis, besides its cost is lower. Therefore it can be developed as a new and potent fibrinolytic medicine. Saccharomyces cerevisiae is recognized as a kind of simple single-cell eukaryotes, which has no toxin and no pathogenicity. It has been used in bread and alcohol industry for years and was affirmed by FDA to be a kind of biological safety.The genomic of Bacillus subticis Var. natto 1.1086 was extracted and nattokinase (NK) gene was amplified by PCR and was linked and transformed. The positive plasmid pMD18T-NK was screened. Sequence analysis showed that the NK gene was 1192 bp in length including signal region, pre-leading region and mature region fragments. The purpose gene had 95％and 69％homology with that of accession No. AY219901.1 from Bacillus subtilis and accession No. DQ178658.1 from Douchi bacteria reported in NCBI, respectively. The homology was much different. It indicated that we can take further study of the differences about nattokinase gene between Bacillus subtilis and douchi bacteria which lead to different functions.The recombinant expression vector pYES2-NK was transformed into Saccharomyces cerevisiae H158 competent cell. The recombinant H158-NK was induced withβ-galactose. The fibrinolytic activity of fermentation supernatant was determined with fibrin plate.The recombinant H158-NK was constructed successfully brings new ideas to develop a new route to construct the efficient genetic transformation of Saccaromyces cerevisiae system to obtain nattokinase with food as bioreactor. Meanwhile, it has set the foundation for developing cheap edible NK products.