A Fibrinolytic Enzyme Produced by &lt;em&gt;Bacillus subtilis&lt;/em&gt; Using Chickpea (&lt;em&gt;Cicer arietinum&lt;/em&gt; L.) as Substrate

Yanping Wang,Rizwan-Ur-Rehman Rizwan-Ur-Rehman,Ping Xiao,Ran Kang,Siping Yao

doi:10.19026/ajfst.6.199

Abstract

A Fibrinolytic Enzyme (BSFE) was isolated from fermented chickpeas using Bacillus subtilis. BSFE was purified with ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The fibrin (ogen) olytic activity of BSFE was investigated by means of fibrinolysis plate and hydrolysis of fibrinogen. Through these steps, the purity of the enzyme increased with 74.60-fold with 6.88% recovery activity. The molecular weight of the BSFE was estimated to be 30 kDa by SDS-PAGE. The optimum pH, optimum temperature, pH stability and thermal stability of BSFE were measured, respectively as 8.0, 55°C, 6.0-8.0 and less than 45°C. The activity was inhibited by serine protease inhibitor PMSF as well as metalloprotease inhibitor EDTA, indicating that the BSFE is a serine metalloprotease. In fibrin plate assay, BSFE showed more stronger fibrinolytic activity than that of nattokinase and it specifically hydrolyzed Aα and Bβ chains followed by γ chain of fibrinogen. Therefore, this study provided a method and it for the preparation of multifunctional food of chickpeas which has strong fibrinolytic activity.

Highlights

Different enzymes from various microbial, animal and plant sources have been popularly used in wide ranges of physiological, medicinal and industrial applications
Plasmin is a “directacting” thrombolytic agent that had been considered to contain greater thrombolytic efficacy and safety than can be afforded by Plasminogen Activators (PA), but it is ineffective when administered by the intravenous route because it is neutralized by plasma α2-antiplasmin (Marder, 2009; Choi et al, 2013)
SDS-PAGE of the purified enzyme was performed in order to verify the enzyme purity and to determine the molecular weight

Summary

INTRODUCTION

Different enzymes from various microbial, animal and plant sources have been popularly used in wide ranges of physiological, medicinal and industrial applications. Intravascular thrombosis due to accumulation of fibrin in arteries is one of the major causes of CVDs. Based on the different working mechanisms, the thrombolytic agents are classified into two types: Plasminogen Activators (PA) and plasmin-like proteins (Peng et al, 2005). In 1897 nattokinase was extracted from natto (Sumi et al, 1987), from on much more attention was paid to the development of new types of fibrinolytic enzyme from food sources such as Korean chungkook-jang soy sauce (Kim et al, 1996), Korean doen-jang (Kim and Choi, 2000), Chinese douchi (Peng et al, 2003) and Asian fermented shrimp paste (Wong and Mine, 2004). Considering the production cost and improvement of enzyme activity, utilizing Bacillus subtilis as a microbial alternative for chickpeas fermentation for the production of fibrinolytic enzyme seems to provide a promising approach. Liquid fermentation of chickpeas with a newly isolated Bacillus subtilis was investigated for the first time and the fibrinloytic enzyme purification, biochemical characterization and thrombolysis ability were evaluated

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION