Selection And Identification Of Strains Producing Fibrinolytic Enzyme From Lobster Sauce And Stuides On Expression Of Fibrinolytic Enzyme Gene In Eukarya

Lobster sauce is a traditional Chinese fermented-soybean food, which has been used for more than one thousand. The fibrinolytic enzyme which is extracted from lobster sauce, has the same action with nattokinase in thrombolysis. Nattokinase is a fibrinolytic enzyme that is extracted from natto, a traditional fermented fool of Japan. The fibrinolytic enztme which is extracted from lobster sauce has stronger thrombolysis activity, longer half life, lower cost, and is safer, more direct and more persistent effect on thrombosis, compared with traditional thrombilytic agents which have been used in common such as streptokinase(SK), urokinase(UK). It also can be used as the preventive medicine of heart and brain vessel disease. All these virtue make it a promising second generation theombolysis medicine.In this research, a bacterial strain producing fibrinolytic enzyme was successfully isolatedefrom lobster sauce which was produced in Fu Shun Town, Si Chuan Province by culture and fibrinolytic activity assay. It was identified as Bacillus subtilis by shape observing, biochemical and physiological characters analysis, and 16S rDNA sequence analysis. It wad named as D10. 16S rDNA sequence suggested that it has 1453bp, and the content of G and C is 55％. The number in GenBank is EF474343.Chromosome DNA was isolated from screened Bacillus subtilis DC10. The fibrinolytic enzyme gene was amplified by PCR. The gene was cloned into Saccharomyces cerevisiae/E. coli shuttle vecter pYES2. Sequncing suggested that the gene of fibrinolytic enzyme is 1158bp. The number in GenBank is EF474344. Sequence homologous analysis displayed thet there were 98％and 93％of nucleotide and amino acid homology between the fibrinolytic enzyme gene and nattokinase gene which reports in NCBI, respectively. So these results suggested that the fibrinolytic enzyme extracted from lobster sauce and nattokinase have high homology.The recombinant expression vector pYES2-DFE (Douchi Fibrinolytic Enzyme) was transformed into Saccharomyces cerevisiae H158 competent cell. Under the induction ofβ-galactose, the fibrinolytic enzyme was expressed.