Extracellular Production of a Potent and Chemically Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology

Background: Nattokinase is a potent fibrinolytic protease, which is used as a drug for treatment or a supplement for preventing thrombosis besides various industrial applications. The present study aimed to produce a soluble nattokinase in low cost media, which has with high activity and resistance to metal ions, detergents, and organic solvents, and can be easily used in medicines or as detergents. Methods: Generally, most of the native extracellular proteins, such as nattokinase from Bacillus subtilis, are lysed by secretory proteases. One way for solving this problem is to employ other hosts for nattokinase production. For producing secretory form of nattokinase from Bacillus subtilis, different factors such as a suitable host, optimized media for the maximum enzyme activity and easy purification are important. Results: These factors are studied in this investigation. Escherichia coli BL21 (DE3), as a reliable host was selected for a high yield production of an extracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b by which the host was transformed. The recombinant nattokinase was expressed through induction with IPTG. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Different media components were evaluated for maximum nattokinase production and activity. The highest enzyme activity of 883.107 U/ml was obtained, when a medium approximately consisting of yeast extract (4.38 g/L), tryptone (4 g/L), K2HPO4 (1.61 g/L) and CaCl2 (0.01 g/L) was used. Conclusion: Entrapping the transformed host in calcium alginate could lead to more enzyme activity and decrease media cost.