3.3 Enhancing hLYZ production by the periodic DO control strategy
After cell concentration reached a relatively high density at the end of
cell growth phase, the successive methanol induction phase was initiated
by feeding methanol to induce heterogeneous protein
synthesis/production. As shown in Fig.3, cell concentration was kept at
an almost constant level throughout the entire methanol induction phase
when using the same methanol induction control strategy of controlling
methanol concentration at 5-10 g L-1. As a result,
achievement of high cell-density cultivation at the end of cell growth
phase (beginning of the methanol induction phase) was an important
factor for efficient heterologous protein production in the successive
methanol induction phase. As shown in Fig.3, when using the periodic DO
control strategy, final hLYZ activity reached the highest level of
2.18×105 IU mL-1 with a higher cell
concentration (~120 g-DCW L-1) at the
end of cells growth phase (Fig.3). This activity was about 2-fold of
that obtained with the “high DO-low glycerol concentration”/DO-stat
control strategy when using the same methanol induction strategy. In the
case of using the “low DO-high glycerol concentration” control
strategy, final hLYZ activity finished at very low levels of
4.71×103 IU mL-1 and the duration of
methanol induction phase was only at about 20 h due to the extremely low
oxygen uptake rate (OUR) and the stoppage of utilizing methanol (Fig.3).
In addition, SDS-PAGE analysis using different glycerol feeding
strategies was also carried out to confirmed above results. As shown in
Fig.3C, the hLYZ protein band obtained with the periodic DO control
strategy (strategy C) were much intensive than those of the other two
batches (strategy A and B).