Nattokinase Bacillus subtilis Ferment: Reduces Blood Viscosity and Fibrinogen Levels in Preclinical Hemorheology Studies

Physicochemical Properties

English Name: NattoKinase
Structural Type: Single-chain polypeptide, alkaline protease

1. Stability of NattoKinase
   pH Stability: NattoKinase is relatively stable under natural pH conditions. It remains stable within 10 minutes at room temperature when the pH rises from 7 to 12. However, it becomes highly unstable and rapidly loses activity when the pH drops below 5. At temperatures exceeding 60°C, it rapidly inactivates due to protein denaturation. After five freeze-thaw cycles, its activity still retains over 95%, indicating that freeze-thawing has minimal impact on NattoKinase activity. When NattoKinase is mixed with boiled wheat extract, boiled meat broth, serum protein, and gastric mucin protein, its stability significantly increases, and even at acidic pH values, the enzyme activity does not completely diminish.

Thermal Stability: It remains relatively stable at temperatures below 45°C and rapidly inactivates due to protein denaturation above 60°C. Freeze-thawing has minimal impact on NattoKinase activity, as it retains over 95% of its activity after five freeze-thaw cycles.

Metal Ion Stability: The activity of NattoKinase is also influenced by certain metal ions. Hg²⁺ completely inactivates it, while Cu²⁺, Zn²⁺, Al³⁺, and Ca²⁺ ions exhibit varying degrees of inhibition. In contrast, Mg²⁺ and Co²⁺ ions activate it.

Stability with Other Substances: E-ACA and EDTA have no significant effect on NattoKinase activity, whereas PMSF completely inhibits it. 1 mmol/L DFP and neguvon also completely inhibit NattoKinase activity.

1. Molecular Weight
   NattoKinase is a single-chain polypeptide enzyme, and the measured molecular weight varies significantly depending on the technique used. Using Sephadex G100 column gel filtration, the result is 20,000, while circular dichroism yields 27,300. Calculating based on the amino acid sequence provides an accurate molecular weight of 27,728.

2. pI Value (Isoelectric Point)
   Using Svunsson's column electrophoresis method and isoelectric focusing, NattoKinase exhibits a symmetrical, single fibrinolytic enzyme peak with a pI value of 8.6 ± 0.3. The isoelectric point is useful for adjusting the pH near the pI value during purification, facilitating enzyme purification.

3. Substrate Specificity of NattoKinase
   As a serine protease, NK's amidolytic activity can be observed using various synthetic substrates. The most sensitive substrate for NattoKinase is S2251, a substrate for plasmin. It also shows activity with substrates H2DLArgPNA, S2238, S2266, and S2302, but is notably inactive with S2444 and S2484. NattoKinase has specific amino acid action and recognition sites for hydrolyzing proteins.

Origin and Application History

Natto is an ancient traditional food, originating in China during the Qin and Han dynasties and introduced to Japan by Zen monks around the Tang dynasty, with a history spanning over a thousand years. Natto, a traditional fermented soybean product in Japan, is made by fermenting cooked soybeans with Bacillus natto under specific temperature and humidity conditions. As a staple side dish in Japan for over two thousand years, it has been used both medicinally and as food. Mature natto has a golden color, a sticky surface coating, and produces long, stringy substances when picked up. With a unique flavor and rich nutrition, it is hailed as a "super health food" and is deeply loved by the Japanese. Currently, natto has become a popular health food in Japan, Canada, the United States, and some European countries, with Chinese enterprises also beginning to produce and sell it.

During the fermentation process of natto, Bacillus natto produces a serine protease (NattoKinase), first discovered in 1987 by Hiroyuki Sumi and colleagues at Miyazaki Medical University in Japan.

Nutritional Components of Natto
During the production of natto, the fermentation process breaks down soybean proteins, increasing the digestibility of natto from the original 50% to 85%, making it more easily absorbed. Natto has high nutritional value, with higher levels of protein, fiber, calcium, iron, vitamins B, PP, and E compared to cooked soybeans, particularly vitamin B, which is over six times higher. Natto also contains proteases, various vitamins, γ-glutamyl transpeptidase, γ-polyglutamic acid, and other substances, as well as high levels of active substances such as NattoKinase, isoflavones, soybean phospholipids, glucosidase, superoxide dismutase (SOD), saponins, tocopherols, pyridine-2,6-dicarboxylic acid, and vitamin K2.