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.
The e#ects on blood coagulation of dipicolinic acid (DPA, ,,0-pyridinedicarboxylic acid), an antibacterial substance known to be produced by Bacillus subtilis natto and contained in natto, a traditional Japanese fermented soybean food, were studied. It was found that addition of DPA with a final concentration of /῍+* ῌ-M results in substantial inhibition of platelet aggregation. DPA inhibition was found to be far stronger than that resulting from addition of aspirin. Furthermore, the clotting reaction of thrombinfibrinogen was also found to be inhibited by DPA. It was confirmed by examination of thromboelastogram patterns that the coagulation of whole rat blood was completely inhibited by addition of /῍+* ῌ-M DPA. From the point of view of the blood coagulation system, these results show that DPA contained in natto may be e#ective in the prevention of thrombosis.
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.
Vitamin K is essential for osteogenesis, and vitamin K deficiency causes osteoporosis, leading to hip fracture and coronary disease. Menaquinone‐7 (MK‐7), one of vitamin K subtypes, is abundantly contained in Japanese traditional fermented food natto. Bacillus subtilis natto secretes a water‐soluble macromolecular complex consisting of MK‐7 and peptides (natto‐MK‐7), although MK‐7 itself is water‐insoluble. However, structural properties of natto‐MK‐7 have not been well elucidated. In the present study, natto‐MK‐7 was purified from a liquid culture medium of Bacillus subtilis natto by fast protein liquid chromatography. The protein assay and electrophoresis suggest that natto‐MK‐7 is a complex composed of three components; menaquinone‐7, 10‐kDa peptide, and 3‐kDa amphiphilic peptides. The hydrodynamic radius of natto‐MK‐7 determined by the dynamic light scattering experiment suggests that natto‐MK‐7 has a more compact shape than that predicted in the previous size‐exclusion chromatography. Practical applications Japanese traditional fermented food natto contains extremely large amount of menaquinone‐7 (natto‐MK‐7): a subtype of vitamin K2. Actually, Japanese natto eaters get about a half amount of vitamin K required daily from natto, and menaquinone‐7 from natto is already on the market. Natto‐MK‐7 is water‐soluble molecular complex, while every type of vitamin K including menaquinone‐7 is insoluble to water. This solubility to water is expected to have an advantage in food/medical applications. The present study is the first report of structural analysis of natto‐MK‐7 by scattering technique. The structural information described in our report can explain how natto‐MK‐7 acquire a water‐solubility, and furthermore, it would be useful for the further improvement of the vitamin K carrier for supplement, pharmaceuticals, and food additives.
Abstracts: It was found that when dipicolinic acid is added to the culture solution of Bacillus subtilis natto, the area of fibrin dissolved by nattokinase (standard fibrin plate) and the amidase activity of nattokinase against Suc-Ala-Ala-Pro-PhepNA increased. For example, when manufacturing natto using steamed soybeans, the addition of 10-64 mM of dipicolinic acid increases amidase activity by more than 10 times.The concentration of vitamin K 2 (menaquinone-7) also increased by about 4 times with the addition of 10 mM of dipicolinic acid. No other food contains such a high concentration of vitamin K 2 . The results were the same for the shaking culture as well as for the stationary culture. If the concentration of dipicolinic acid is appropriately controlled, a product with excellent levels for both nattokinase activity and vitamin K 2 concentration could be manufactured.
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