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\begin{document}

\title{High cell density cultivation of a recombinant Bacillus subtilis for
nattokinase production}



\author[1]{Qing Cui}%
\author[1]{Bingjun Qian}%
\author[1]{Xiangjun Sun}%
\author[1]{Jianhua Zhang}%
\affil[1]{Shanghai Jiao Tong University}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Several hundred U mL-1 of Nattokinase (NK), a fibrinolytic enzyme, can
be produced by culturing recombinant Bacillus subtilis in Luria-Bertani
broth in a shaking flask. For use as a nutraceutical, large-scale
preparation and a simple purification process is required. The present
study utilized a fed-batch process with a pH-stat and
low-glycerol-level-maintain feeding strategy to cultivate a B. subtilis
strain carrying a pHT01 plasmid with an NK-encoding gene (B.
subtilis/pHT01-aprN1). Finally, a NK activity of 7778 \selectlanguage{ngerman}±17.28 U mL-1 was
obtained, which represented a 26-fold increase of NK activity by high
cell density cultivation compared to the flask culture. Furthermore,
fermentation supernatant was successively purified by ammonium sulfate
precipitation and nickel column affinity chromatography with a total NK
recovery rate of 65.2\%.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Keywords}

Nattokinase; \emph{Bacillus subtilis} ; recombinant protein expression;
high cell density fermentation; induction and feeding strategy

\textbf{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 \emph{B. subtilis} 168 strain containing a
pHT01-\emph{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.

\textbf{Materials and Methods}

\textbf{Microorganism and inoculum preparation}

A gene-modified strain (\emph{B. subtilis}168/pHT01-\emph{aprN1} ) was
used to express NK. The medium for flask and fed-batch culture was as
follows: yeast extract, 60 g L\textsuperscript{-1}; glucose, 40 g
L\textsuperscript{-1}; glycerol, 20 mL L\textsuperscript{-1};
NH\textsubscript{4}Cl, 3 g L\textsuperscript{-1};
K\textsubscript{2}HPO\textsubscript{4}\selectlanguage{ngerman}·3H\textsubscript{2}O, 1 g
L\textsuperscript{-1}; MgSO\textsubscript{4}·7H\textsubscript{2}O, 2 g
L\textsuperscript{-1}; MnSO\textsubscript{4}·H\textsubscript{2}O, 0.02 g
L\textsuperscript{-1}; FeSO\textsubscript{4}·7H\textsubscript{2}O, 0.05
g L\textsuperscript{-1}; CoCl\textsubscript{2}·6H\textsubscript{2}O,
0.01 g L\textsuperscript{-1}; ZnCl\textsubscript{2}, 0.01 g
L\textsuperscript{-1}; 2,6-pyridinedicarboxylic acid, 0.167 g
L\textsuperscript{-1}. Glucose and
MgSO\textsubscript{4}·7H\textsubscript{2}O were autoclaved separately
and aseptically added to the medium. Nutrient solution contained 100 g
L\textsuperscript{-1} yeast extract and 250 mL
L\textsuperscript{-1}glycerol was used as feed medium. To avoid plasmid
loss and prevent microbial contamination, chloramphenicol (5 \selectlanguage{greek}μ\selectlanguage{english}g
mL\textsuperscript{-1}) was added to all media after being dissolved in
anhydrous ethanol and filtered through a 0.22 \selectlanguage{greek}μ\selectlanguage{english}m membrane.

\textbf{Shake flask experiments}

To prepare a stock culture, one colony of \emph{B.
subtilis}168/pHT01-\emph{aprN1} was inoculated in 1 mL Luria-Bertani
(LB) medium and then incubated overnight at 37\selectlanguage{ngerman}°C in a shaking incubator
at 200 revolutions per minute (rpm). A flask with 25 mL fed-batch
culture media was then inoculated with 7.5\% (v/v) of stock culture and
incubated at 37°C until reaching mid-log phase (OD\textsubscript{600} of
0.8--1.0). Next, the temperature was adjusted to 28°C and held for 20
min, after which a 25-\selectlanguage{greek}μ\selectlanguage{english}L aliquot of 0.5 mmol L\textsuperscript{-1}
isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG) was added and the culture
was further incubated for 45 h at 28\selectlanguage{ngerman}°C in a shaking incubator (200 rpm)
for NK expression.

\textbf{Fed-batch experiments}

An in-situ sterilizable 15 L bioreactor (BIOSTAT\textsuperscript{®}B,
Germany) equipped with pH and pO\textsubscript{2} probes was used for
fed-batch experiments. One milliliter of glycerol stock bacteria was
inoculated into 100 mL LB medium and cultured for 12 h at 37°C in a
shaking incubator (200 rpm), after which the pre-culture solution was
aseptically inoculated into 1 L LB medium and incubated under the same
conditions for 12 h. Next, 700 ml of seed cultures were inoculated for
7-L fed-batch fermentation in the bioreactor. The value of oxygen
dissolved was set at 20\%, which was cascade controlled by agitation
speed (300--1000 rpm) and air flow rate (3--20 L
min\textsuperscript{-1}). The pH was set as 7.0 and automatically
controlled by adding 2 mol L\textsuperscript{-1} HCl or 2 mol
L\textsuperscript{-1} NaOH.

In the first fed-batch experiment (FB A), the temperature was adjusted
to 28°C at 3.5 h, while IPTG was added 30 min later as the
OD\textsubscript{600} value reached about 18. A total of 600 mL feed
media was supplemented at 8 h with a flow rate of 30 mL
min\textsuperscript{-1}. In the second fed-batch experiment (FB B), the
expression was induced at 5 h when the OD\textsubscript{600} value had
reached 27, and the feed media was constantly fed after 14 h of
cultivation with a flow rate of 6 mL min\textsuperscript{-1} for 1 h,
then 3.6 mL min\textsuperscript{-1} for 2 h, and finally 2.4 mL
min\textsuperscript{-1} for another hour. In the third experiment (FB
C), the expression was induced at 3 h when the OD\textsubscript{600}
reached about 9 and the feed media was fed at 10 h. The feeding rate
depended on the content of glycerol, which was controlled at a
concentration of 50 mmol L\textsuperscript{-1}. Cell growth was
monitored at various times by measuring the OD\textsubscript{600}
values.

\textbf{NK activity assay}

Quantitative analysis of NK activity was conducted by the fibrin plate
method, with slight modification {[}17{]}. Briefly, bovine fibrinogen
(Sigma, St. Louis, MO, USA) and thrombin (Sigma, St. Louis, MO, USA)
were dissolved in 0.1 mol L\textsuperscript{-1} sodium phosphate buffer
(PBS) at pH 7.4. An equi-voluminal mixture of 5 g L\textsuperscript{-1}
bovine fibrinogen solution and 12 g L\textsuperscript{-1} agarose
solution was then warmed in a 45°C bath, after which 10 \selectlanguage{greek}μ\selectlanguage{english}L of 500 U
thrombin was added to 15 mL of the mixture solution in a 90 mm petri
dish and kept at room temperature for 1 h to form fibrin. Holes with a 2
mm diameter were made on the fibrin plate and 10 \selectlanguage{greek}µ\selectlanguage{english}L of each diluted
supernatant of fermentation broth was then added. The plates were
subsequently incubated at 25\selectlanguage{ngerman}°C for 16 h, after which the areas of the
lysis zones on the fibrin plates were measured and the fibrinolytic
activities were determined according to the standard curve of urokinase.

\textbf{Glycerol concentration measurement}

The concentration of glycerol was determined enzymatically using a free
glycerol assay kit (Sigma, St. Louis, MO, USA) according to the
manufacturer's procedures.

\textbf{Purification and}

\textbf{lyophilization of NK}

The final fermentation broth was collected and centrifuged (GL-25M,
Luxiangyi, Shanghai, China) immediately at 18,300 × g for 10 min. Solid
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4} was then
gradually added until the supernatant was 20\% saturated, after which it
was stored at 4°C overnight. Next, the broth was centrifuged at 18,300 ×
g for 10 min and the precipitate was discarded. Additional
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4} was
subsequently added to make the supernatant 60\% saturated, after which
the mixture was stored at 4°C overnight again. Following centrifugation
at 18,300 × g for 30 min, the supernatant was removed and the
precipitate was dissolved in 10 mmol L\textsuperscript{-1} PBS (pH 7.4)
as a crude enzyme. The crude enzyme was further purified by Ni--NTA
affinity chromatography according to the manufacturer's (Novagen, San
Diego, CA, USA) protocols. After purification, 10 \selectlanguage{greek}µ\selectlanguage{english}l of eluted fractions
of different dilution times were used to assay their fibrinolytic
activities. Residual solution was desalted by dialysis and lyophilized
using a GLZY-0.5B vacuum freeze drier (Pudong Freeze Drying Equipment,
Shanghai, China) to obtain NK Lyophilized powder.

\subsubsection*{SDS-PAGE and Western blotting
analysis}

{\label{sds-page-and-western-blotting-analysis}}

SDS-PAGE protein analysis was performed in a Mini-Protean Tetra system
(BioRad, Hercules, CA, USA) by loading 30 \selectlanguage{greek}μ\selectlanguage{english}L of 1: 1 (v: v) boiled
supernatant and dye buffer onto a 4--20\% precast Mini protean TGX gel
(BioRad) and then running the samples in 1 \selectlanguage{ngerman}× Tris-glycine-SDS running
buffer (BioRad, Shanghai, China) at 100 V for 15 min followed by 200 V
for 25 min. A protein standard (10-250 kDa, Precision Plus Protein
Kaleidoscope, Bio-Rad) was used as a ladder.

NK was identified by Western blotting analysis as described by Towbin,
with slight modification {[}18{]}. Briefly, 10 \selectlanguage{greek}μ\selectlanguage{english}L aliquots of different
supernatants were separated by 15\% SDS--PAGE. After electrophoresis,
the protein bands were transferred from the gel to an Amersham
nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA) using
a Mini Trans Blot electrophoretic transfer cell (BioRad). NK on the
membrane was reacted with a mouse monoclonal antibody (Yisheng
Biological Technology, Shanghai, China) against His-tag (1:1000 v/v),
then incubated with anti-mouse \emph{Ig} G alkaline phosphatase (Sigma)
conjugate (1:2000 v/v). After washing, the NC membrane was stained using
Super ECL Detection Reagent (Yisheng Biology Technology, Shanghai,
China) according to the manufacturer's instructions, then covered by an
X-ray film and exposed for 1 min. A Tanon v.3500 Gel Imaging System
(Tanon Co., Shanghai, China) was used for the analysis of proteins.

\textbf{Results and discussion}

\textbf{NK expression by flask culture and fed-batch experiment}

At least five different NK activity assay methods have been reported to
date {[}4, 11, 12, 17, 19,{]}. Among these, the fibrin plate method and
chromogenic method are the most widely used, but the casein-degradation
and JBSL (Japan Bio Science Laboratory Co., Ltd) methods may show much
higher values of enzyme activity {[}11, 12, 14{]}.

The NK activity obtained at 24 h in flask culture was 380.14 \selectlanguage{ngerman}± 5.71 U
mL\textsuperscript{-1}, while the OD\textsubscript{600} reached 13.45 ±
0.45.

Many studies have demonstrated the significant contribution of media
ingredients {[}20, 21{]} and nutrient feeding strategy to NK production
{[}4, 15{]}. Berenjian confirmed that glycerol was a noteworthy carbon
source influencing cell density during the fermentation of \emph{B.
subtilis} natto, and that the highest activity of NK was obtained by
adding 3\% glycerol as a carbon source {[}15{]}. It has also been
reported that 2,6-pyridine dicarboxylic acid (PDCA) and metal ions such
as Ca\textsuperscript{2+} and Mg\textsuperscript{2+} could improve
osmotic pressure and help to maintain enzyme conformation, thereby
improving the NK activity {[}10, 22{]}. Moreover, Wang demonstrated that
glucose, K\textsubscript{2}HPO\textsubscript{4·}3H\textsubscript{2}O and
MgSO\textsubscript{4}·7H\textsubscript{2}O played key roles in the
production of NK, and they obtained an activity of 12.34 FU
mL\textsuperscript{-1} {[}23{]}. Taken together, the results of these
studies showed that NK activity could be improved dozens of times by
media optimization.

In addition to media, feeding solutions are critical factors that
influence NK activity, which should support cell growth and recombinant
protein production while avoiding substrate inhibition and other related
problems {[}12, 15{]}.

Based on this information, we selected a mixture of glycerol, yeast
extract, PDCA and a concentrated inorganic mixture solution as the
fermentation broth, and a mixture of glycerol and yeast extract as the
feed broth.

The strategy of induction, including cell density at the time of
induction, inducer concentration, pre-induction growth and
post-induction incubation time, can also affect the efficiency of
protein expression. The aim of this study was to investigate the effects
of using pH-stat and low-glycerol-level-maintaining strategies on NK
expression by \emph{B. subtilis} 168/pHT01-\emph{apr} N1.

Three experiments were performed to examine the effects of induction
time, feeding time and feeding rate on the NK activity of fermentation
broth. The results are presented in Figure 1 and Table 1.

For FB A, the OD\textsubscript{600} increased rapidly from the second
hour\emph{.} Although we fed 600 mL of media into the fermenter at the
eighth hour, the nutrition was not sufficient for cell growth as
indicated by the glycerol concentration decreasing rapidly to about 70
mmol L\textsuperscript{-1} from 4 to 12 h and the
OD\textsubscript{600}not varying markedly after 13 h. However, the NK
activity still increased significantly until 17 h. The final NK activity
was 2910.5 ± 21.6 U mL\textsuperscript{-1} and the specific activity was
30.32 U ml\textsuperscript{-1}
OD\textsubscript{600}\textsuperscript{-1}.

For FB B, we attempted to achieve higher enzyme production via higher
cell density; hence, the expression was induced at the fifth hour, which
was 1 h later than FB A, and the induced OD\textsubscript{600} was up to
27.3 ± 1.0, which was higher than that of FB A (17.6 ± 0.4). The feeding
time of FB B was 14 h, when the glycerol concentration was as low as
119.8 ± 1.3 mmol L\textsuperscript{-1}, which might have favored cell
growth by reducing substrate inhibition. The glycerol content was
sufficient to support the cell growth for 24 h, and the
OD\textsubscript{600} reached a high value of 208.8 ± 1.9 at 24 h, with
the highest activity reaching 4521.8 ± 23.8 U mL\textsuperscript{-1}.
Interestingly, the specific activity was 21.66 U
ml\textsuperscript{-1}OD\textsubscript{600}\textsuperscript{-1} at 24 h,
which was lower than that of FB A, and the NK activity did not
synchronously increase following cell growth during the late
fermentation stage, which implied that there should be a balance between
the cell growth rate and enzyme expression. Consequently, we did not
unilaterally pursue high cell density in FBC, and instead induced at an
earlier time and kept the glycerol content low during the feeding period
in FB C.

For FB C, continuous feeding was adapted, starting at 10 h when the
OD\textsubscript{600} had reached 105.3 ± 1.1 and almost two-thirds of
the initial glycerol had been consumed. The glycerol concentration was
controlled to around 50 mmol L\textsuperscript{-1} by adjusting the
feeding rate.

As expected, an activity of 7778.0 ± 17.3 U mL\textsuperscript{-1} and a
specific activity of 44.86 U
ml\textsuperscript{-1}OD\textsubscript{600}\textsuperscript{-1} was
achieved at 20 h, and these values were 1.7-, 2.6-, and 26-fold higher
than those of batch B, batch A and the \selectlanguage{english}flask culture, respectively. These
values were also higher than those of other studies in which the
reported NK production levels were 587 U mL\textsuperscript{-1}
{[}15{]}, 7100 U mL\textsuperscript{-1} {[}12{]}, and 3194.3 U
mL\textsuperscript{-1}{[}4{]}, respectively.

\textbf{Recovery rate of NK by purification}

The recovery rate of NK obtained by ammonium sulfate precipitation was
89.1\% (Table 2), which was consistent with a study by Garg {[}16{]}.
The high recovery rate and simple operation showed that this method
could be used to purify NK on a large scale.

The purification was followed by Ni--NTA affinity chromatography, and
the total recovery rate was 65.2\% (Table 2). The high imidazole
concentration in wash buffer B led to a high loss of NK when samples
were purified using Ni--NTA affinity chromatography. Accordingly, the
imidazole concentrations were adjusted to zero in wash buffer A and
incubated supernatant, and to 10 mmol L\textsuperscript{-1} in wash
buffer B; thus, a recovery rate of 88\% was obtained in this step.
Different imidazole concentrations in elution buffer C were also
investigated, but there were no significant differences in the range of
100 mmol L\textsuperscript{-1} to 500 mmol L\textsuperscript{-1}.
According to the NK expression level and the recovery rate, the process
developed here may be applied for large scale production of NK.

\textbf{SDS-PAGE and Western blotting analysis of NK}

SDS-PAGE analysis demonstrated that a 28 kDa protein was a crucial
component in the supernatant from induced \emph{B.
subtilis}168/pHT01-\emph{apr} N1\emph{,} but that it was not present in
the supernatant from \emph{B. subtilis} 168/pHT01 and non-induced
\emph{B. subtilis} 168/pHT01-\emph{apr} N1 fermentation broth (Fig. 2
A/B), suggesting that recombinant NK could be expressed in a soluble
form. Western blotting with a His-tag-specific monoclonal antibody also
showed a specific signal at 28 kDa, whereas no cross-reaction occurred
in the total soluble proteins from non-induced \emph{B. subtilis}
168/pHT01-\emph{apr} N1 broth\emph{,}which confirmed that the 28 kDa
protein was the recombinant NK, as expected (Fig. 2 C).

In view of these reports, NK was produced by fed-batch cultures of
recombinant \emph{B. subtilis} , and its production was improved to
7,778 U/mL from 380 U/mL of flask culture using pH-stat and
low-glycerol-level strategies. Future studies will design a process and
set a kinetic model for fermentation optimization.

\textbf{Acknowledgment}

This project was supported by the National Key Research and Development
Program of China (No. 2017YFD0400205).

\textbf{Conflict of interest}

The authors have no conflicts of interest to declare.

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