Extrachromosomal expression of nat05 gene encoding an alkaline serine protease from Bacillus subtilis N05

Abstract

Nattokinase, the fibrinolytic serine protease, has been shown to be beneficial in preventing strokes caused due to blood clots. Therefore, synthesis of recombinant nattokinase in a host system, which can be both safe for human consumption as well as demonstrate good productivity and amenable to the downstream processing, will have a great socioeconomic significance. A nat05 gene encoding nattokinase – the most important serine protease secreted by the Bacillus subtilis natto – was cloned from B. subtilis strain N05 and expressed using pHT43 plasmid in another B. subtilis strain (BD170). Enzymatic assays, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram, were employed to assess the efficiency of the production of nattokinase using a relatively novel recombinant DNA technique. Among the five recombinant bacterial clones (R1–R5), R4 was found to secrete high amounts of nattokinase into the culture medium, as evidenced by the total proteolytic enzyme assay, fibrinolytic enzyme assay, SDS-PAGE, and zymogram. A preliminary analysis of the biochemical properties of the crude enzyme showed characteristics that are typical of nattokinase as reported by other studies. Thus, in this study, we were able to successfully produce nattokinase in B. subtilis strain (BD170) using recombinant DNA approach. The R4 recombinant clone that was found to secrete nattokinase can be used as a source of recombinant protein that can be further purified for various therapeutic applications, or the strain could be used as a probiotic bacterium in functional foods.

Keywords

• <i>Bacillus subtilis </i>N05
• fibrinolytic activity
• nattokinase
• alkaline serine protease