Statistical optimization of fibrinolytic enzyme production using agroresidues by Bacillus cereus IND1 and its thrombolytic activity in vitro.

Ponnuswamy Vijayaraghavan,Samuel Gnana Prakash Vincent

doi:10.1155/2014/725064

Ponnuswamy Vijayaraghavan, Samuel Gnana Prakash Vincent

Open Access

https://doi.org/10.1155/2014/725064

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Abstract

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.

Highlights

Cardiovascular diseases (CVDs) including acute myocardial infarction, ischemic heart disease, peripheral vascular disease, high blood pressure, and stroke are the leading causes of death worldwide [1]
The results showed that fibrinolytic enzyme production by B. cereus IND1 varied with the type of substrates
A potent fibrinolytic enzyme was produced using agroresidues by B. cereus IND1. This organism effectively utilized wheat bran for the fibrinolytic enzyme production, and the process parameters were optimized by two-level full-factorial design and the Response surface methodology (RSM)

Summary

Introduction

Cardiovascular diseases (CVDs) including acute myocardial infarction, ischemic heart disease, peripheral vascular disease, high blood pressure, and stroke are the leading causes of death worldwide [1]. Fibrinolytic enzymes can be found in a variety of foods, such as Japanese natto, tofuyo, Korean cheonggukjang soy sauce, edible honey mushroom [1], Chinese douche [5], Indonesian tempeh [6], Taiwanese fermented red bean [7], Japanese shiokara [8], and Asian fermented shrimp paste [9]. These fibrinolytic enzymes possess three antithrombotic activities, which included the conversion of plasminogen to plasmin, the activation of t-PA, and the degradation of fibrin by fibrinolytic activity of plasmin in conjunction with nattokinase [10]

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