The Optimization, Extraction And Purification Of Nattokinase And Genetic Cloning

Cardiovascular and cerebrovascular diseases have"high incidence, high morbidity, high mortality, high complications"and other features. In 2005, the world health organization(WHO) indicated that about 1,700 people died of cardiovascular and cerebrovascular diseases annually[1]. With the improvement of human life and the pressure increases, the trend is getting younger and younger. As the most deadly reason, thrombotic prevention is a top priority. Although traditional medicines have significant effect on the prevention and therapies of cardiovascular and cerebrovascular diseases and thromboembolic disease, but have serious side effects; As the development and use of preventive health care functional food, traditional fermented soybean food make a ignificant and effective security .According to historical records, natto fermented soybean originated in China, through the introduction of Buddhism from China to Japan. Long life expectancy in addition to the natural environment, the more important is their dietary structure, especially the consumption of fermented food natto, considered the biggest secret of Japanese lo-ngevity, as a national treasure food. Soybean protein content as high as 35.3%, enzymes from the natto production can make 50% of the protein into a water-soluble, digest up to 80%. It also contains amino acids, vitamins, vegetable fat, etc. According to scientific research shows that natto food is not only significant gastrointestinal function adjustment, but also has treatment of colds, diarrhea, flatulence, gastroenteritis, good digestion, residual stools, constipation and other effects, as well as diabetes control, inhibition of thrombus formation and dissolution of thrombus, which is a kind of longevity, health, beauty foods.In 1987, Sumi Dr. for the first time isolated the role of a strong fibrinolytic alkaline protease from traditional food natto and named Nattokinase in Japanese. It could directly cross-link fibrin, but also activate plasminogen in vivo, thus demonstrating a strong thrombolytic effect. It is reported that their products have many functions,for example, hydrolysising of amyloid, decreasing factor VII and factor VIII of plasma fibrinogen, lowering blood viscosity, blood fat, cholesterol, blood pressure, improving blood circulation state, maintenancing normal blood cell morphology and function, improving the regeneration of nerve injury , inhibiting of osteoporosis, atherosclerosis and other functions]. Since NK has good security, low cost, rapided into the blood after oral administration, stability of the gastrointestinal tract, by bacterial fermentation product, also produced by genetically engineered bacteria, etc. It is expected to be developed as a new generation of oral Antithrombotic drugs for thrombotic disease prevention and treatment. Nattokinase mainly from Japan, and many people can not accept its unique flavor. Although nattokinase capsules has been producted in Japan, South Korea, North Korea, but only short-term supply and high cost, its application is limited. To make it convenient and widely used, people has done further research from the perspective of molecular biology to find a better expression vector and host strain, and optimization of expression conditions.Objectives In this study, We used traditional methods nattokinase extraction and purification to detecte its activity through the optimization of fermentation broth, salt, chromatography, protein content, fibrinolytic activity assay, SDS-PAGE and other techniques and tools. We extracted natto bacillus subtilis genomic DNA, designed primers, PCR cloning nattokinase through molecular biology techniques. Select the appropriate expression vector and host bacteria to apply more secure products, high-quality purified thrombolytic drugs, in order to provide the theoretical basis for clinical application.Methods 1. In this study, We measured OD600 value by continuous turbidimetry[1], drawed the growth curve of bacteria, identified Bacillus natto culture conditions, in order to better applied in practical production. To further improve the enzyme production, reduce fermentation costs, we intend to use the single factor and orthogonal experiment to optimize the fermentation process.2. We centrifugated the crude enzyme solution, salted out 40% to 45%. CM-Cellulose ion exchange chromatography and Sephadex G-100 gel column can be extracted out of a single enzyme. Then, we determinated molecular weight by SDS-PAGE, compared of crude enzyme solution and extracted a single enzyme's activity.3. We extracted natto bacillus subtilis genomic DNA , PCR amplify the sequence, and detected by 1% agarose gel electrophoresis. The study remains to be further studied of construction of recombinant plasmid and transformed into Esherichia coli.Results We centrifugated the crude enzyme solution, salted out 40% to 45%. CM-Cellulose ion exchange chromatography and Sephadex G-100 gel column can be extracted out of a single enzyme. To reduce the loss of enzyme during the operation, the whole process strived at 4℃, operated under sterile conditions. Then, We recycled the cloned products. When we selected the appropriate expression vector and host bacteria, the repeated expression was not successful. However, the method provided in this experiment was worth learning to researchers.Conclusions 1. The optimal combination of medium composition is MgSO4 0.02%, K2HPO4 0.02%, KH2PO4 0.01%, CaCl2 0.05%, Maltose 3%, Peptone of soybean 3%. The optimum temperature is 37℃, and the optimum pH is 8, also the best inoculum for each 5g soybean inoculation 500μL. The nattokinase production of optimizating fermentation conditions, increased from 31.25IU/ml to 158.74IU/ml. The unit of production has gone up about 5.08.2. Nattokinase Extracted through the CM-Cellulose ion exchange chromatography, contained active ingredient in 0.10 mol/L NaCl elution peak, and Folin-phenol method to measure the enzyme activity was 168.56 IU/ml, but Sephadex G-100 gel filtration chromatography to measure the enzyme activity was 137.16 IU/mL, and their molecular mass was 28 000 Da determined by SDS-PAGE.3. Bacillus subtilis natto extracted from genomic DNA, amplificated by PCR(275 amino acids, bases 825bp) and detected nattokinase original sequence (381 amino acids, bases 1143bp), and confirmed by 1% agarose gel electrophoresis.