Keywords
Nattokinase; Bacillus subtilis ; recombinant protein expression; high cell density fermentation; induction and feeding strategy
Introduction
Thrombosis, which is responsible for high morbidity and mortality in humans [1], can be effectively treated by thrombolytic drugs, but these are associated with adverse effects [2, 3]. Therefore, it is necessary to develop new biological substances, especially prophylactic food-source thrombolytic agents with low immunogenicity and preventative, long-term effects that are convenient for oral administration and stable in the gastrointestinal tract.
Nattokinase (NK), which decreases the ability of blood to clot, is traditionally taken from natto, a Japanese solid-state fermented soybean food [5, 6]. Currently, NK is used as a dietary supplement as well as a prophylactic or a curative thrombosis medicine [4, 5]; however, the process used to purify it from natto is complicated and accompanied by significant loss of bioactivity. Accordingly, fed-batch fermentation by adopting genetically modified bacteria may improve enzyme yield [7-9].
Several reports have shown that fed-batch culture led to increases in NK activity of 2.1–25-fold relative to batch culture and that the addition of glycerol during the cell growth phase increased NK production significantly [11–15]. Moreover, various protein purification methods have been utilized for NK purification [4, 16]. Taken together, these findings suggest that the expression of NK by genetically modified bacteria may reach a much higher level if efficient protocols for high cell density fermentation and subsequent purification are obtained.
We previously constructed a B. subtilis 168 strain containing a pHT01-aprN plasmid [10]. In this study, we further investigated the expression of NK by culturing this strain under different induction and feeding strategies. In addition, we investigated the efficiency of purification with ammonium sulfate precipitation and Ni–NTA affinity chromatography.