3.2. Effect of PhaP location on PHB-binding activity
For the proposed approach to be a valid option for enzyme immobilization, the PHB-binding activity of phasin fusion proteins should be carefully considered alongside the enzymatic activity of the target enzyme. Because the hydrophobic region of its C-terminal end needs to be exposed for PhaP to interact with PHA (Zhao et al. 2016), we presumed that it would be more favorable to place a fusion partner in in front of phasin. This presumption was confirmed by observation of PHB nanofibers under fluorescence microscopy after incubation with sfGFP-PhaP or PhaP-sfGFP. As expected, when PHB nanofibers were incubated with the same molar amount of fusion proteins, much greater fluorescence was observed from those incubated with sfGFP-PhaP (Figure 2). By comparison, binding of PhaP-sfGFP to PHB nanofibers was only marginally higher than the control sfGFP.
In accordance with this result, the amount of LipM7-PhaP on PHB nanofibers was almost twice that of PhaP-LipM7 (Figure 3A). Furthermore, time-course analysis revealed an exceptionally stable maintenance of LipM7-PhaP on PHB nanofibers. As shown in Figure 3B, upon storage in buffer solution, >70% of immobilized LipM7-PhaP remained on PHB nanofibers after 28 days. By comparison, wild-type LipM7 adsorbed onto PHB nanofibers was almost completely released into buffer within a week. These results clearly indicate that C-terminal fusion of PhaP is desirable for maintaining the enzymatic activity of LipM7 and the binding activity of PhaP. LipM7-PhaP was therefore used in subsequent experiments.