Nattokinase

Subtilisin NAT
Crystal structure of nattokinase. PDB 4dww.
Identifiers
OrganismBacillus subtilis natto
SymbolaprN
OrthologsOMA: entry
UniProtP35835
Other data
EC number3.4.21.62
Search for
StructuresSwiss-model
DomainsInterPro

Nattokinase (pronounced nah-TOH-kin-ayss) is an enzyme originally extracted from a Japanese food called nattō. Nattō is produced by fermentation by adding the bacterium Bacillus subtilis var. natto, which also produces the enzyme, to boiled soybeans. In spite of its name, nattokinase is not a kinase enzyme (and should not be pronounced as such, i.e. nah-toh-KYN-ayss), but an extracellular serine protease of the subtilisin family (99.5% identical with subtilsin E). Rather, it is named for the fact that it is an enzyme produced by nattōkin (Japanese: 納豆 菌).

This enzyme is labeled subtilisin NAT (S08.044) in the MEROPS database. Synonyms include nattokinase, subtilisin QK, subtilisin QK02. The gene encodes a prepropeptide with a signal peptide and an inhibitor propeptide before the main enzyme chain (residues 107–381, theoretic molecular mass 27726.74). It has the designation Bac s 1 under the WHO/IUIS allergen nomenclature.

Catalytic activity

In vitro

When in contact with human blood or blood clots, it exhibits a strong fibrinolytic activity. It can both cleave fibrin and inactivate plasminogen activator inhibitor-1 (PAI-1).

Fibrinolytic unit

The fibrinolytic unit (FU) is a measure of fibrinolytic activity used in the labeling of nattokinase supplements. It specifically measures the rate of tyrosine liberation in a solution of fibrin.

In vivo

Intravenous route

Nattokinase is able to dissolve blood clots when injected intravenously into rats.

Oral route

Although nattokinase should be digested and inactivated in the human gut like other proteins, one group reported that it has some effect when consumed orally.

Two studies examined the fate of nattokinase in the gut:

  • A 1995 article reported that after rats received nattokinase injected into the duodenum, specific bands show up when blood serum is analyzed using Western blot and an anti-NK antibody. One band occurs at the same location as control nattokinase (28 kDa).
  • A 2013 article reported that after ingestion, nattokinase can be detected in blood using enzyme-linked immunosorbent assay (ELISA). However, this is not sufficient proof that nattokinase is absorbed in a functional form, as ELISA frequently cross-reacts with digested versions of an antigen.

A 2018 review stated that convincing data is "available to demonstrate the bioavailability and metabolism of NK administered as an oral dose".

A double-blind randomized controlled trial published in 2021 reported that in "healthy individuals at low risk for CVD", nattokinase has no effect on carotid artery intima-media thickness and carotid arterial stiffness, two measures of subclinical atherosclerosis progression.

Production

Nattokinase used to be extracted from nattō. The same molecule can now be produced more efficiently by recombinant means and in batch culture.

Many legume products are fermented using B. subtilis (or a close relative). Many contain an identical or near-identical protease with the ability to dissolve fibrin in vitro. These enzymes are referred to as nattokinase in some academic sources.

Culture

The Japan Nattokinase Administration, an industry interest group, runs a certification program for nattokinase products that are derived only from natural B. subtilis var. natto. It considers all other sources "counterfeit".

See also

References

  1. ↑ Yanagisawa Y, Chatake T, Chiba-Kamoshida K, Naito S, Ohsugi T, Sumi H, et al. (December 2010). "Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto". Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 66 (Pt 12): 1670–73. Bibcode:2010AcCrF..66.1670Y. doi:10.1107/S1744309110043137. PMC 2998380. PMID 21139221.
  2. ↑ "MEROPS - the Peptidase Database". www.ebi.ac.uk.
  3. ↑ "Expasy - PeptideMass". web.expasy.org.
  4. ↑ "Bac s 1 Allergen Details". allergen.org.
  5. ↑ Urano T, Ihara H, Umemura K, Suzuki Y, Oike M, Akita S, et al. (July 2001). "The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1". The Journal of Biological Chemistry. 276 (27): 24690–96. doi:10.1074/jbc.M101751200. PMID 11325965.
  6. ↑ Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S (December 1993). "Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan". Biochemical and Biophysical Research Communications. 197 (3): 1340–47. Bibcode:1993BBRC..197.1340F. doi:10.1006/bbrc.1993.2624. PMID 8280151.
  7. ↑ "Can You Take Nattokinase With Coumadin (Warfarin)?". PharmacistAnswers. Retrieved 2019-02-20.
  8. ↑ Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (October 1987). "A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet". Experientia. 43 (10): 1110–11. Bibcode:1987Expea..43.1110S. doi:10.1007/bf01956052. PMID 3478223. S2CID 13476341.
  9. ↑ S, MK; V, M; M, M; C, SD (8 July 2025). "Hyperproduction of nattokinase from Bacillus subtilis VIT MS2 using random mutagenesis and statistical optimization through central composite design". BMC Microbiology. 25 (1): 424. doi:10.1186/s12866-025-04150-w. PMC 12235895. PMID 40629276. The reaction mixture consisted of 100 µL of enzyme solution, pre-incubated with 300 µL of 100 mM Tris-HCl buffer (pH 7.4) containing 10 mM CaCl₂ for 5 min at room temperature. About 300 µL of 1.2% (w/v) fibrin solution was added, and the mixture was incubated at 37 °C for 10 min. [...] One fibrinolytic unit (FU) was defined as the amount of enzyme required to release 1 µg of tyrosine per minute under assay conditions/
  10. ↑ Fujita, Mitsugu; Hong, Kyongsu; Ito, Yae; Fujii, Rie; Kariya, Kimio; Nishimuro, Satoshi (1995). "Thrombolytic Effect of Nattokinase on a Chemically Induced Thrombosis Model in Rat". Biological and Pharmaceutical Bulletin. 18 (10): 1387–1391. doi:10.1248/bpb.18.1387. PMID 8593442.
  11. ↑ Chen H, McGowan EM, Ren N, Lal S, Nassif N, Shad-Kaneez F, et al. (2018). "Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases". Biomarker Insights. 13 1177271918785130. doi:10.1177/1177271918785130. PMC 6043915. PMID 30013308.
  12. ↑ Fujita, Mitsugu; Hong, Kyongsu; Ito, Yae; Misawa, Satoru; Takeuchi, Naoto; Kariya, Kimio; Nishimuro, Satoshi (1995). "Transport of Nattokinase across the Rat Intestinal Tract". Biological and Pharmaceutical Bulletin. 18 (9): 1194–1196. doi:10.1248/bpb.18.1194. PMID 8845803.
  13. ↑ Ero, MP; Ng, CM; Mihailovski, T; Harvey, NR; Lewis, BH (May 2013). "A pilot study on the serum pharmacokinetics of nattokinase in humans following a single, oral, daily dose". Alternative Therapies in Health and Medicine. 19 (3): 16–9. PMID 23709455.
  14. ↑ Leder, L; Wendt, H; Schwab, C; Jelesarov, I; Bornhauser, S; Ackermann, F; Bosshard, HR (15 January 1994). "Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides". European Journal of Biochemistry. 219 (1–2): 73–81. doi:10.1111/j.1432-1033.1994.tb19916.x. PMID 8307037.
  15. ↑ Chen, H; McGowan, EM; Ren, N; Lal, S; Nassif, N; Shad-Kaneez, F; Qu, X; Lin, Y (2018). "Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases". Biomarker Insights. 13 1177271918785130. doi:10.1177/1177271918785130. PMC 6043915. PMID 30013308.
  16. ↑ Hodis, HN; Mack, WJ; Meiselman, HJ; Kalra, V; Liebman, H; Hwang-Levine, J; Dustin, L; Kono, N; Mert, M; Wenby, RB; Huesca, E; Rochanda, L; Li, Y; Yan, M; St John, JA; Whitfield, L (2021). "Nattokinase atherothrombotic prevention study: A randomized controlled trial". Clinical Hemorheology and Microcirculation. 78 (4): 339–353. doi:10.3233/CH-211147. PMID 33843667.
  17. ↑ Liang X, Jia S, Sun Y, Chen M, Chen X, Zhong J, Huan L (November 2007). "Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli". Molecular Biotechnology. 37 (3): 187–94. doi:10.1007/s12033-007-0060-y. PMID 17952663. S2CID 11595829.
  18. ↑ Li X, Wang X, Xiong S, Zhang J, Cai L, Yang Y (October 2007). "Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells". Biotechnology Letters. 29 (10): 1459–64. doi:10.1007/s10529-007-9426-2. PMID 17581705. S2CID 26608552.
  19. ↑ Cho YH, Song JY, Kim KM, Kim MK, Lee IY, Kim SB, et al. (September 2010). "Production of nattokinase by batch and fed-batch culture of Bacillus subtilis". New Biotechnology. 27 (4): 341–46. doi:10.1016/j.nbt.2010.06.003. PMID 20541632.
  20. ↑ Kwon EY, Kim KM, Kim MK, Lee IY, Kim BS (September 2011). "Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis". Bioprocess and Biosystems Engineering. 34 (7): 789–93. doi:10.1007/s00449-011-0527-x. PMID 21336955. S2CID 38816306.
  21. ↑ Keziah, S. Merlyn; Devi, C. Subathra (December 2021). "Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata". The Protein Journal. 40 (6): 876–890. doi:10.1007/s10930-021-10023-8. PMID 34611797.
  22. ↑ Association, Japan NattoKinase. "JNKA Mark". Japan NattoKinase Association (in Japanese).