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.