Purification and characterization of recombinant nattokinase from Bacillus subtilis R0H1


  • Nguyen Thi Thuy Ngan School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet Road, Hai Ba Trung District, Hanoi, Vietnam
  • Le Tuan School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet Road, Hai Ba Trung District, Hanoi, Vietnam
  • Nguyen Lan Huong School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet Road, Hai Ba Trung District, Hanoi, Vietnam




Bacillus subtilis, characterization, nattokinase, purification, recombinant


Nattokinase (NK) is a fibrinolytic enzyme with the potential for fighting cardiovascular diseases (CVD) thanks to its antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and neuroprotective effects. Nattokinase was first discovered and purified from soybean fermented food by Bacillus subtilis natto. To enhance NK’s activity and simplify downstream processes, production of recombinant NK using several microbial expression systems such as Escherichia coli, B. subtilis, and Lactococcus lactic has been studied. Among all of them, B. subtilis is a prominent host for overproduction of functional proteins which can be secreted directly into the culture medium. In this study, recombinant NK from B. subtilis R0H1 was purified using two-step membrane filtration. Results showed 3.2-fold increase in activity and a recovery rate of more than 80%. Molecular weight of NK was approximately 28 kDa and its fibrinolytic degradation capacity was proved according to SDS-PAGE. The optimal pH and temperature of this NK were 8.5 and 55°C, respectively. The enzyme activity was boosted by Mg2+, Ca2+ and obviously inhibited by Co2+, Zn+2, Fe2+, and SDS. The apparent Km and Vmax with fibrin as the substrate were 3.08 mM and 6.7 nmol/min, respectively. The results suggested that membrane filtration is a useful method for purification of recombinant NK from B. subtilis R0H1. Therefore, application of membrane system is proposed to purify NK at the pilot scale. In addition, our findings indicated that recombinant NK produced in B. subtilis R0H1 showed high and stable proteolytic activity in slightly alkaline pH and at high temperature. It also exhibited strong fibrinolytic activity again both substrates: fibrinogen and fibrin.


Download data is not yet available.


Bora B, Gogoi D, Tripathy D, Kurkalang S, Ramani S, Chatterjee A, Mukherjee AK (2018) The N-terminal-truncated recombinant fibrin(ogen)olytic serine protease improves its functional property, demonstrates in vivo anticoagulant and plasma defibrinogenation activity as well as pre-clinical safety in rodent model. International Journal of Biological Macromolecules 111: 462-474.

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1): 248-254.

Chang CT, Wang PM, Hung YF, Chung YC (2012) Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chemistry 133: 1611–1617.

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 13:1-8.

Cui W, Suo F, Cheng J, Han L, Hao W, Gua J, Zhou Z (2018) Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis. Microbial biotechnology 11(5): 930-942.

Deepak V, Ilangovan S, Sampathkumar MV, Victoria MJ, Pasha SPBS, Pandian SBRK, Gurunathan S (2010) Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles. Enzyme and Microbial Technology 47: 297-304.

Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimoro S (1995) Thrombolytic effect of nattokinase on a chemically Induced thrombosis model in Rat. Biological & Pharmaceutical Bulletin 18(10): 1387-1391.

Garg R, Thorat BN (2014) Nattokinase purification by three phase partitioning and impact of t-butanol on freeze drying. Separation and Purification Technology 131: 19-26.

Hmood SA, and Aziz GM (2016) Purification and characterization of nattokinase produced by local isolate of Bacillus sp. B24. Iraqi Journal of Biotechnology 15(2): 93-108.

Hu Y, Yu D, Wang Z, Hou J, Tyagi R, Liang Y, Hu Y(2019) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food. Scientific Reports 9(1): 9235.

Kaptoge, Pennells S, Bacquer LD, Cooney D, Kavousi MT, Stevens M, Riley G, Savin LM, Khan S, Altay T, Amouyel S, et al (2019) World Health Organization cardiovascular disease risk charts: revised models to estimate risk in 21 global regions. The Lancet Global Health 7(10): 1332-1345.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the Head of Bacteriophage T4. Nature 227(5259): 680-685.

Liang X, Zhang L, Zhong J, Huan L (2007) Secretory expression of a heterologous nattokinase in Lactococcus lactis. Appl Microbiol Biotechnol 75(1): 95-101.

Lin HTV, Wu GJ, Hsieh MC, Chang SH, Tsa GJ (2015) Purification and characterization of nattokinase from cultural filtrate of red alga porphyra dentata fermented by Bacillus subtilis N1. Journal of Marine Science and Technology 23: 240-248.

Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. Journal of the American Chemical Society 56(3): 658-666.

Liu Z, Zheng W, Ge C, Cui W, Zhou L, Zhou Z (2019) High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters. BMC Microbiology 19: 89.

Reuß DR, Schuldes J, Daniel R, Altenbuchner J (2015) Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain 3NA. Genome announcements 3(2).

Ren Y, Pan X, Lyu Q, Liu W (2018) Biochemical characterization of a fibrinolytic enzyme composed of multiple fragments. Acta Biochim Biophys Sin 50(2): 227-229.

Sumi H, Hamada H, Nakanishi K, Hiratani H (1990) Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematologica 84(3): 139-143.

Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43(10): 1110-1111.

Thao NT, Quyen DT, and Hoang LT (2013) Cloning and enhancing production of a detergent and organic solvent resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease gene deficient Bacillus subtilis WB800. Microbial Cell Factories, 12: 79.

Tian L, Zhou W, Zhang Y (2019) Construction of a genetically engineered strain of nattokinase and assessment of its fibrinolytic activity. African Journal of Microbiology Research 13(279): 488-499.

Tuan TQ, Ai LTT, Hiep DM, Dong TC (2015) Purification and characterization of recombinant nattokinase from Bacillus subtilis. Tạp chí sinh học 37: 75-84.

Walker JB, Nesheim ME (1999) The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. Journal of Biological Chemistry 274(8): 5201-5212.

Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y (2009) Purification and characterization of nattokinase from Bacillus subtilis natto B-12. Journal of Agricultural and Food Chemistry 57(20): 9722-9729.

Wang SL, Wu YY, Liang TW (2011) Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. New Biotechnology 28(2): 196-202.

Wu DJ, Lin CS, Lee MY (2009) Lipid-lowering effect of nattokinase in patients with primary hypercholesterolemia. Acta. Cardiol. Sin. 25: 26-30.

Xiao-Lan L, Lian-xiang D, Fu-Ping L, Xi-Qun Z, and Jing X (2005). Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Applied Microbiology and Biotechnology 67(2): 209-214.

Xin X, Ambati RR, Cai Z, Lei B (2018) Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. 3 Biotech 8(2): 90

Xin X, Ambati RR, Cai Z, Lei B (2019) Development of universal purification protocols for fibrinolytic enzyme-producing bacilli. CyTA - Journal of Food 17(1): 112-120.

Yongjin C, Wei B, Shujun J, Meizhi W, Yan J, Yan Y, Zhongliang Z, Guolin Z (2011) Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto. FEMS Microbiol Lett 325(2): 155-161.




How to Cite

Thuy Ngan, N. T., Tuan, L., & Huong, N. L. (2022). Purification and characterization of recombinant nattokinase from Bacillus subtilis R0H1. Vietnam Journal of Biotechnology, 20(2), 369–377. https://doi.org/10.15625/1811-4989/16027