Abstract
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 D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O 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.
Highlights
Natto is a Japanese traditional fermented food made from soybeans, and is considered a health-food product in Japan, because it is rich in proteins and valuable chemical compounds such as nattokinase (NK; Sumi et al, 1987) and vitamin K2 (Sakano et al, 1988)
Previous mass spectroscopic analysis indicated this lot reached 100% in six successive cultivations and the the possibility of glycosylation or other chemical reactions final Bacillus subtilis natto Miyagino (BSNM) optimized for deuteration (BSNM–D2O) could be (Chiba-Kamoshida et al, 2010); no modification was obtained after a further two cultivations in 100% D2O
While the results indicated that D2O strongly inhibited growth and NK production of the native BSNM, BSNM–D2O had acquired resistance to D2O
Summary
Natto is a Japanese traditional fermented food made from soybeans, and is considered a health-food product in Japan, because it is rich in proteins and valuable chemical compounds such as nattokinase (NK; Sumi et al, 1987) and vitamin K2 (Sakano et al, 1988). NK is produced in abundance by the fermentation of soybeans using Bacillus subtilis natto and extracted to medium by sporulation. The fibrinolytic activity derived from NK is enhanced in tissues. These previous reports indicate the potential for medical usage of NK, as in the case of therapeutic and diagnostic agent for thrombosis. NK is a serine protease composed of 275 amino acids (molecular weight 27.7 kDa) and belongs to the subtilisin family; Sumi and co-workers found that the
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