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.