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.

Key words： nattokinase (NK); Bacillussp.; clot busters; docking

Acknowledgements

The authors acknowledge the management of VIT University for providing all the necessary facilities to carry out the study.

Compliance with ethics guidelines

Mohan, Priya, Swaroop, Sourav, Srinath, Sweta., Merlyn & Subathra declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors.

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