In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

Abstract

BACKGROUND: Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from  Bacillus  sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation.

METHODS: In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed.

RESULTS: Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding.

CONCLUSIONS: A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

Keywords

nattokinase (NK) / Bacillussp. / clot busters / docking

References

[1] Bryan P N (2000). Protein engineering of subtilisin. Biochim Biophys Acta, 1543(2): 203–222

[2] Chang C T, Fan M H, Kuo F C, Sung H Y (2000). Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem, 48(8): 3210–3216

[3] Chen P T, Chiang C J, Chao Y P (2007). Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis. Biotechnol Prog, 23(4): 808–813

[4] Darnell S J, LeGault L, Mitchell J C (2008). KFC Server: interactive forecasting of protein interaction hot spots. Nucleic Acids Res, 36(Web Server issue): W265-9

[5] Venkataraman Deepak, Suresh Babu, Ram Kumar Pandian, KalimuthuKalishwaralal, Sangiliyandi Guru Nathan (2009). Purification, Immobilization, and Characterization of NK on PHB Nanoparticles. Elsevier. Bioresour Technol, 100: 6644–6646

[6] Dominguez C, Boelens R, Bonvin A M (2003). HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc, 125(7): 1731–1737

[7] Dubey R, Kumar J, Agrawala D, Char T, Pusp P (2011). Isolation, Production, Purification, Assay and Characterization of Fibrinolytic Enzymes. Afr J Biotechnol, 10(8): 1408–1420

[8] Holmstrom B (1965). Streptokinase assay on large agar diffusion plates. Acta Chem. Isolation, production, purification, assay and characterization of fibrinolytic enzymes (NK, Streptokinase and Urokinase) from bacterial sources. Afr J Biotechnol, 10(8): 1408–1420

[9] Laemmli U K (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680–685

[10] Matta H, Punj V. 1998. Isolation and partial characterization of a thermostable extracellular protease of Bacillus polymyxa B-17. Internat J Food Microbiol, 42: 139–145

[11] Mohanasrinivasan V, Subathra Devi C, Ritusree Biswas, Falguni Paul, MohorMitra, Selvarajan E (2013). Enhanced production of NK from UV mutated Bacillus sp. Bangladesh J Pharm, 1991–0088

[12] Motaal A A, Fahmy I, El-Halawany A, Ibrahim N (2015). Comparative fibrinolytic activities of nattokinases from Bacillus subtilis var. natto. Pharm Sci Res, 7(2): 63–66

[13] San-Lang H C, Liang T W, Lin Y D (2009). A Novel NK Produced By pseuodomonas sp. TKU015 Using Shrimp Shells As substrate, Elsevier, 2008. Process Biochem, 44: 70–76

[14] Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark A E, Berendsen H J C (2005). GROMACS: fast, flexible, and free. J Comput Chem, 26(16): 1701–1718

[15] Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y (2009). Purification and characterization of nattokinase from Bacillus subtilis natto B-12. J Agric Food Chem, 57(20): 9722–9729

[16] Wu S M, Feng C, Zhong J, Huan L D (2011). Enhanced production of recombinant nattokinase in Bacillus subtilis by promoter optimization. World J Microbiol Biotechnol, 27(1): 99–106

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg