3.3.2 Production of rrBChE
The total active rrBChE during the induction phase is shown in Figure
3c. The culture medium rrBChE was significant compared with the
cell-associated rrBChE starting at 2 dpi. The active rrBChE levels in
the medium and cell extract at 5 dpi were 23.0 ± 3.8 µg/ g FW and 61.6 ±
0.6 µg/ g FW, respectively, corresponding to total active rrBChE of 84.6
± 13.8 µg/ g FW (Table 2), which is at least 1.5-fold higher than the
semicontinuous cycles previously described in Section 3.1.3 but
comparable to the results acquired from the lab-scale study at 5 dpi
(Macharoen et al., 2020). The accumulation of total active rrBChE may or
may not increase after 5 dpi; however, keeping rice cells longer in the
induction phase may lead to a decrease in viable cell density (Figure
S1b), longer recovery times, and may not increase rrBChE productivities.
Figure 3d shows the increase of cell-associated rrBChE purity in the
induction phase due to the increase of cell-associated rrBChE production
level (Figure 1c) and the decrease of TSP (data not shown). This rrBChE
purity pattern is similar to the lab-scale study (Macharoen et al.,
2020) . The maximum purity of cell-associated rrBChE of 1.87 ± 0.26% g
rrBChE/g TSP (Table 2) was at least 2-fold greater than cycles 1-4 in
the semicontinuous run but 1.25-fold lower than previous study
(Macharoen et al., 2020), whereas the maximum purity of culture medium
rrBChE of 0.55 ± 0.02% g rrBChE/g TSP was at most 1.5-times lower than
cycles 2-4 in the semicontinuous run. Overall, the maximum total active
rrBChE concentration and maximum cell-associated rrBChE purity was
significantly improved in this run compared with the semicontinuous run.
Figure 4 shows a Western blot analysis under reducing conditions of
rrBChE samples during the induction phase. Crude cell extracts from 0-5
dpi were loaded into lanes 1-6, respectively, with an equal volume of 20
µL. As a result, the rrBChE band intensity represents the concentration
of combined active and inactive cell-associated rrBChE when the
extraction ratio was maintained at 1 g FW per 1mL extraction buffer. The
rrBChE bands at 0 and 1 dpi (lanes 1 and 2, respectively) were not
visible due to the low concentrations of rrBChE (~4 and
~12 ng active rrBChE/µL, respectively), while the
intensity of rrBChE bands increased, as expected, from 2 dpi to 5 dpi
(lanes 3-6, respectively) indicating the increase of rrBChE
concentrations in the crude extract. Lane 7, under reducing condition,
represents hBChE derived from blood plasma as a positive control showing
the majority of monomeric hBChE (around 85 kDa) and a small fraction of
dimeric hBChE. Lane 8 was loaded with 30 µL of 45X concentrated rrBChE
from the culture medium showing slightly different molecular weight
compared to the control and rrBChE from crude extract. Comparing with
monomeric hBChE, the monomeric rrBChE bands from both crude extract and
concentrated medium appear slightly below 85 kDa may be due to fewer
occupied N -glycosylation sites in rrBChE resulting in smallerN -glycan structures (Corbin et al., 2018; Kolarich et al., 2008).
The rrBChE bands at 5 dpi from the crude extract (lane 6) and the
culture medium (lane 8) show different molecular weights probably due to
different N -glycoforms suggesting that rrBChE in the culture
medium is most likely secreted to the rice cell wall and then the medium
via the secretory pathway.
The maximum volumetric and specific productivities of total active
rrBChE in this 13-day batch cultivation were 387± 66 µg
L-1 day-1 and 76.1 ± 13.2 µg g
DW-1 day-1 (Table 2), respectively,
resulting in 1.4-fold and 1.8-fold, respectively, greater productivities
than what obtained in cycles 3-4 in the semicontinuous run. The
different rrBChE production levels and productivities between the
semicontinuous run and the batch run was probably caused by different
hydrodynamic stress (with and without baffles in the semicontinuous run
and the batch run, respectively) and overall mass transfer. It could be
interpreted that maintaining kLa as a scaling-up
parameter in a bioreactor and eliminating baffles improves rrBChE
productivities and allows comparable results between the lab-scale
bioreactor and pilot scale bioreactor using the similar bioreactor
conditions (Macharoen et al., 2020). By replicating the growth kinetics
and rrBChE production kinetics in the pilot-scale bioreactor as
previously found in the lab-scale bioreactor, we are successful in
scaling up the production of rrBChE using kLa as a
scaling-up parameter.