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 ± 5.71 U
mL-1, while the OD600 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 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 Ca2+ and Mg2+ could improve
osmotic pressure and help to maintain enzyme conformation, thereby
improving the NK activity [10, 22]. Moreover, Wang demonstrated that
glucose, K2HPO4·3H2O and
MgSO4·7H2O played key roles in the
production of NK, and they obtained an activity of 12.34 FU
mL-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 B. subtilis 168/pHT01-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 OD600 increased rapidly from the second
hour. 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-1 from 4 to 12 h and the OD600not 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-1 and the specific activity was 30.32 U
ml-1 OD600-1.
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 OD600 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-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
OD600 reached a high value of 208.8 ± 1.9 at 24 h, with
the highest activity reaching 4521.8 ± 23.8 U mL-1.
Interestingly, the specific activity was 21.66 U ml-1OD600-1 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 OD600 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-1 by adjusting the feeding rate.
As expected, an activity of 7778.0 ± 17.3 U mL-1 and a
specific activity of 44.86 U ml-1OD600-1 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 flask culture, respectively. These values were also
higher than those of other studies in which the reported NK production
levels were 587 U mL-1 [15], 7100 U
mL-1 [12], and 3194.3 U mL-1[4], respectively.