Screening, Identification And Mutagenesis Breeding Of Plasmin-producing Strains In Soybean Paste

Thrombotic diseases are the leading causes of death throughout the world.A tremendous amount of research on searching for natural thrombolysis material has been done in the area of prevention and the treatment of the diseases.Microorganisms producing fibrinolytic material have widely been found in a variety of fermentation food,such as Japanese Natto,Chinese Douchi and Korean Chungkook-Jang soy sauce.These fibrinolysis materials have significant potential for food fortification and pharmaceutical applications,such that their use could effectively prevent and cure thrombotic diseases.A strain producing fibrinolytic enzyme was isolated from soy-bean paste which was sampled from Baiquan county of Qiqihar district by means of fibrin plate.Then a mutant in possession of high fibrinolytic avtivity and genetic stability was achieved by mutagenesis treatment of nitrosoguanidine and He-Ne laser.Analysis on characterization of this fibrinolytic enzyme had been done to investigate its safety,thrombolysis and anticoagulation effect in vitro.A strain producing fibrinolytic enzyme named HDBF-DJ3 was isolated from soy-bean paste by the step of pykno-extracted fibrin plate accumulating and casein plate separating.Its fibrinolytic activity was 183.38±2.45 IU/mL and was initially identified as Bacillus subtilis by testing its morphological,physiological and biochemical characteristics as well as 16S rDNA sequence analysis.A mutant strain HDBF-DJ3N7 was obtained from HDBF-DJ3 by mutagenesis treatment of nitrosoguanidine with its final concentration 0.1 mg/mL and mutagenic time 25 min.The fibrinolytic activity of HDBF-DJ3N7 was 368.78±9.88 IU/mL,which was 2.1 times higher than that of HDBF-DJ3.Then HDBF-DJ3N7 was treated with He-Ne laser(?=632.8nm)with its power 30 mW,vertical length 20 cm and exposure time 45 min.And a mutant strain HDBF-N7HN5 with high yield of fibrinolytic enzyme was obtained,whose fibrinolytic activity was 429.89±5.74 IU/mL,and its enzyme production was 2.3 times higher than that of HDBF-DJ3.Genetic stability analysis on HDBF-N7HN5 indicated that the production of fibrinolytic enzyme was very stable after 10 generation cultivation.Analysis on characterization of this fibrinolytic enzyme showed that it could not only degrade fibrin directly,but also activae plasminogen into plasmin then hydrolyze fibrin indirectly.It had different affinity to the three moity of fibrin,first is the a chain,then is the ? and ? chain last.And this kind of fibrinolytic enzyme was safety for application as well as had the effect of thrombolysis and anticoagulation in vitro.