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Cardiovascular and cerebrovascular diseases, with a high incidence of morbidity, mortality, complications and more \In 2005, the World Health Organization (WHO) survey showed that around the world each year about 17 million people died from cardiovascular and cerebrovascular diseases. With the improvement of the level of human life as well as the pressure of living increase, and showed a trend of younger, thrombosis is the most deadly diseases in the cardiovascular and cerebrovascular diseases. Therefore, prevention and treatment of thrombosis is a priority. Traditional medicines significant effect on cardiovascular and cerebrovascular and thromboembolic disease prevention, but serious side effects; traditional fermented soybean food is not only effective and safe non-toxic and can be used as a prevention and health food development and use of value function . According to historical records, natto, tempeh, which originated in China and Buddhism from China into Japan, Japan's per capita long life reasons in addition to the more excellent natural environment, the more important is the dietary structure, fermented foods, especially consumer The largest amount of natto, is the secret of Japanese longevity, regarded as a national treasure food. The monasteries eating soy because of its protein content as high as 35.3%, while 50% protein can become water-soluble enzyme produced natto, digestibility up to 80%. Also contains amino acids, vitamins, vegetable fat. According to scientific research shows that natto food not only significantly adjust gastrointestinal function, but also has the effect of treatment of colds, diarrhea, flatulence, gastroenteritis, indigestion, residual stools, constipation, as well as diabetes control, inhibition of thrombus formation and dissolution of thrombus The role is a longevity, health beauty food. It can be said that natto is a natural food product of human wisdom. Dr. Sumi Japan for the first time in 1987, isolated from the traditional Japanese food natto, a strong fibrinolytic alkaline protease and named nattokinase (nattokinase, NK). It can not only direct effect of the cross-linked fibrin, but also plasminogen activation in vivo, thus demonstrating a strong role in thrombolytic. Its products have reportedly hydrolysis amyloid lowering plasma fibrinogen, Factor VII and Factor VIII, reduce blood viscosity, lowering blood pressure, lower cholesterol, lower blood pressure, improve blood circulation state, maintain normal morphology and function of blood cells, conducive to the regeneration of nerve injury, and inhibition of osteoporosis, inhibition of atherosclerosis and other multiple functions. Since NK having good security, low cost, and rapidly into the blood after oral administration, and long duration of action, and good stability of the gastrointestinal tract, can be fermented by the bacteria to produce, but also by genetic engineering strain production, etc., is expected to be developed as a new generation of oral antithrombotic drugs for the prevention and treatment of thrombotic diseases. But until now, the source of nattokinase mainly Japanese natto fermentation because of its unique flavor of a lot of people can not accept. Containing nattokinase as a main component of the capsule formulations have been produced in Japan, South Korea, North Korea, but only short-term supply and high price, its application is limited. In order to make it convenient and widely used, people from the perspective of molecular biology to find a better expression vector and host strain, optimization of expression conditions do further research, the application of genetic engineering products purified more quality thrombolytic drugs, have broader application prospects will bring a new dawn to the clinical. The aim of the present study was optimized through fermentation broth salting out, chromatography, protein content, fibrinolytic activity assay technologies and means of SDS-PAGE, extraction and purification of nattokinase through traditional methods to detect its activity. Also by molecular biology techniques, extracting the natto bacillus subtilis genomic DNA, primers were designed, the PCR cloning nattokinase and nattokinase original. Select the appropriate expression of the expression vector and host strain, to the application of genetic engineering products purified a safer quality thrombolytic drugs, provide a theoretical basis for clinical application. Method 1 in this experiment by turbidimetry [1] continuously measured OD600 of strain growth curves to determine natto culture conditions to better applied to actual production. In order to further improve the enzyme yield, reduce fermentation costs, we have adopted the single factor and orthogonal experiment to optimize the fermentation process. The crude enzyme solution by centrifugation, salting out of 40 ~ 45%, CM-Cellulose ion-exchange chromatography and Sephadex G-100 gel column can be extracted from a single enzyme, and determination of molecular weight by SDS-PAGE, the crude enzyme solution and extraction a single enzyme activity of the enzyme solution. 3. Natto Bacillus subtilis genomic DNA extraction, PCR amplification nattokinase and nattokinase original sequence, and a 1% agarose gel electrophoresis detection. For further construction of the recombinant plasmid, and transformed into E. coli pave the way for the follow-up work remains to be further studied. Results 1. The optimal combination of the medium components of MgSO4 0.02% of MgSO4, 0.02%, KH2PO4 0.01%, CaCl2 0.05% maltose, 3%, 3% of soybean peptone, optimal culture temperature of 37 ° C, the optimum pH was 8.0, and most good vaccination 5g soybeans inoculated with 500μL. Nattokinase production optimization of fermentation conditions, enzyme activity increased by optimization the front 31.25IU/mL fermentation liquid to 158.74IU/mL fermentation broth unit increase in production of about 5.08 times. 2 by the CM-Cellulose ion-exchange chromatographic separation nattokinase enzyme to obtain a 0.10 mol / L NaCl elution peak containing the active ingredient, the Folin-phenol method measured activity 168.56 IU / m. To 0.10mol / L NaCl elution peak Nattokinase enzymes were separated by Sephadex G-100 gel filtration chromatography, the enzyme activity measured by Folin-phenol method to 137.16 IU / mL, and they were measured by SDS-PAGE molecular mass 28 000 Da. 3. Natto Bacillus subtilis genomic DNA extraction, PCR amplification of nattokinase (275 amino acids, bases 825bp) and nattokinase original sequence (381 amino acids, bases 1143bp), and 1% agarose gel electrophoresis confirmed. Conclusion after optimization of the liquid medium to extract a crude enzyme solution, centrifuged, and 40 to 45% by salting, CM-Cellulose ion-exchange chromatography and Sephadex G-100 gel column can be extracted from a single enzyme, the enzyme to reduce the operating process losses, the whole process seeks under sterile conditions at 4 ℃, operation. Nattokinase and nattokinase original gene cloning and recycling. Select suitable expression vector and host strain is not successful, repeated expression, with reference to the experimental method available for future scientists, pending further study.
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