Bacillus natto ( Bacillus subtilis natto ) was cultivated, and an analysis was conducted after performing lysozyme treatment and water extraction of the culture supernatant and the B. subtilis natto cells. The intracellular existence of a large amount of water-soluble vitamin K (Menaquinone-7: MK-7) was established. The existence of small amounts of other types of vitamin K2 including MK-4 and PK was also confirmed in the culture solution (watersoluble fractions). The amount of water-soluble menaquinone-7 in Bacillus was 85 m g/g wet weight of the bacteria, and the amount was equivalent to more than 100 times as much as that contained in the culture solution (0.02 m g).Gel filtration using Sephacryl S-200HR revealed that the molecular weight of the water-soluble menaquinone-7 is approximately MW 200,000, and isoelectric focusing revealed a behavior similar to that of protein, with a pI of about 4.2. A rabbit antibody was prepared with this water-soluble vitamin K as the antigen. By using the Ouchterlony method, the antibody showed a reaction (precipitation line) with the water-soluble menaquinone-7 prepared from both the intracellular fraction and the extracellular culture solution, and it was found through the antigen-antibody reaction that the menaquinone-7 disappears from the supernatant of the reactant (the watersoluble menaquinone-7 is thus neutralized). Based on these results, it was inferred that the vitamin K produced by B. subtilis natto becomes watersoluble by forming an intracellular complex with protein and is released in the extracellular fraction during the culture process.
Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å , = 95.2. Diffraction images were processed to a resolution of 1.74 Å with an R merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.
Extraction of nattokinase, a fibrinolytic enzyme in natto bacillus, was attempted by the following 4 methods: 1) extraction with saline, 2) treatment with the organic solvents acetone, toluene and hexane prior to extraction, 3) alkaline treatment at pH 11.0 and, 4) autolysis in the presence of 0.1%NaN 3 at 4°C. Each fraction showed not only a strong fibrinolytic activity, but also H-D-Val-Leu-Lys-pNA and Suc-Ala-Ala-Pro-Phe-pNA amidolytic activities. Doses of 50-200 mg/kg natto bacillus (1؋10 11 active cells/g) were orally administered for rat experimental pulmonary thrombosis and to healthy human volunteers. A decrease in thrombus count and plasma euglobulin lysis time (ELT), as well as an increase in tissue plasminogen activator (t-PA), indicate that natto bacillus serves to activate plasma fibrinolysis in vivo.
Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D 2 O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D 2 O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.
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