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).